U.S. patent number 7,030,155 [Application Number 10/151,066] was granted by the patent office on 2006-04-18 for emulsion vehicle for poorly soluble drugs.
This patent grant is currently assigned to Sonus Pharmaceuticals, Inc.. Invention is credited to Panayiotis P. Constantinides, Karel J. Lambert, Steven C. Quay, Alexander K. Tustian.
United States Patent |
7,030,155 |
Lambert , et al. |
April 18, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Emulsion vehicle for poorly soluble drugs
Abstract
A method of making an emulsion of tocopherol incorporating a
co-solvent and, stabilized by biocompatible surfactants, as a
vehicle or carrier for therapeutic drugs, which is substantially
ethanol free and which can be administered to animals or humans by
various routes, is disclosed. Also included in the emulsion is
PEGylated vitamin E. PEGylated .alpha.-tocopherol includes
polyethylene glycol subunits attached by a succinic acid diester at
the ring hydroxyl of vitamin E and serves as a primary surfactant,
stabilizer and a secondary solvent in emulsions of
.alpha.-tocopherol.
Inventors: |
Lambert; Karel J. (Woodinville,
WA), Constantinides; Panayiotis P. (Bothell, WA),
Tustian; Alexander K. (Bothell, WA), Quay; Steven C.
(Edmonds, WA) |
Assignee: |
Sonus Pharmaceuticals, Inc.
(Bothell, WA)
|
Family
ID: |
26778490 |
Appl.
No.: |
10/151,066 |
Filed: |
May 17, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20030065024 A1 |
Apr 3, 2003 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09317495 |
May 24, 1999 |
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60088269 |
Jun 5, 1998 |
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Current U.S.
Class: |
514/449; 424/455;
424/486 |
Current CPC
Class: |
A61K
9/0019 (20130101); A61K 9/1075 (20130101); A61K
47/22 (20130101) |
Current International
Class: |
A01N
43/16 (20060101) |
Field of
Search: |
;514/449
;424/455,486 |
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|
Primary Examiner: Low; Christopher S. F.
Assistant Examiner: Delacroix-Muirheid; C.
Attorney, Agent or Firm: Christensen O'Connor Johnson
Kindness PLLC
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
09/317,495, filed May 24, 1999, now abandoned, which claims the
benefit of U.S. Provisional Application No. 60/088,269, filed Jun.
5, 1998. Each application is incorporated herein by reference in
its entirety.
Claims
We claim:
1. A method of making an emulsion, comprising: a) dissolving one or
more chemotherapeutic agents in polyethylene glycol to form a
chemotherapeutic agent solution, wherein the chemotherapeutic agent
is at least one of a taxoid, a taxine, a taxane, or a
microtubule-binding agent; b) adding a tocopherol polyethylene
glycol to the chemotherapeutic agent solution to form an oil
solution; and c) adding a tocopherol to the oil solution to form a
tocopherol/chemotherapeutic agent solution.
2. The method of claim 1 further comprising blending the
tocopherol/chemotherapeutic agent solution with an aqueous phase to
form a pre-emulsion.
3. The method of claim 2 further comprising homogenizing the
pre-emulsion to form a fine emulsion.
4. The method of claim 1 wherein the molecular weight of the
polyethylene glycol is in the range from 200 to 600.
5. The method of claim 1, wherein the tocopherol is
.alpha.-tocopherol.
6. The method of claim 1, wherein the tocopherol polyethylene
glycol is an ester or an ether of .alpha.-tocopherol and
polyethylene glycol.
7. The method of claim 1 wherein the tocopherol polyethylene glycol
is d-.alpha.-tocopherol-polyethylene glycol 1000 succinate.
8. The method of claim 1 further comprising adding a
polyoxypropylene-polyoxyethylene glycol nonionic block polymer with
the tocopherol polyethylene glycol to the chemotherapeutic agent
solution.
9. The method of claim 1, wherein the chemotherapeutic agent is
paclitaxel.
10. A method for making a paclitaxel/tocopherol-containing
solution, comprising: a) combining paclitaxel and polyethylene
glycol to provide a first paclitaxel-containing solution; b) adding
a tocopherol polyethylene glycol and a
polyoxypropylene-polyoxyethylene glycol nonionic block polymer to
the first paclitaxel-containing solution to provide a second
paclitaxel-containing solution; and c) adding a tocopherol to the
second paclitaxel-containing solution to provide a
paclitaxel/tocopherol-containing solution.
11. The method of claim 10, wherein the polyethylene glycol has a
molecular weight in the range from 200 to 600.
12. The method of claim 10, wherein the tocopherol polyethylene
glycol comprises d-.alpha.-tocopherol polyethylene glycol 1000
succinate.
13. The method of claim 10, wherein the tocopherol comprises
.alpha.-tocopherol.
14. A method far making a paclitaxel/tocopherol-containing
emulsion, comprising: a) combining paclitaxel and polyethylene
glycol to provide a first paclitaxel-containing solution; b) adding
a tocopherol polyethylene glycol and a
polyoxypropylene-polyoxyethylene glycol nonionic block polymer to
the first paclitaxel-containing solution to provide a second
paclitaxel-containing solution; c) adding a tocopherol to the
second paclitaxel-containing solution to provide a third
paclitaxel-containing solution; d) blending the third
paclitaxel-containing solution with an aqueous phase to form a
pre-emulsion; and e) homogenizing the pre-emulsion to form an
emulsion.
15. The method of claim 14, wherein the polyethylene glycol has a
molecular weight in the range from 200 to 600.
16. The method of claim 14, wherein the tocopherol polyethylene
glycol comprises d-.alpha.-tocopherol polyethylene glycol 1000
succinate.
17. The method of claim 14, wherein the tocopherol comprises
.alpha.-tocopherol.
18. A method for making a paclitaxel/tocopherol-containing
solution, comprising: a) combining paclitaxel and polyethylene
glycol to provide a first paclitaxel-containing solution; b) adding
a tocopherol/polyethylene glycol conjugate to the first
paclitaxel-containing solution to provide a second
paclitaxel-containing solution; and c) adding a tocopherol to the
second paclitaxel-containing solution to provide a third
paclitaxel-containing solution.
19. The method of claim 18, wherein the polyethylene glycol has a
molecular weight between 200 and 600.
20. The method of claim 18, wherein the tocopherol/polyethylene
glycol conjugate comprises d-.alpha.-tocopherol polyethylene glycol
1000 succinate.
21. The method of claim 18, wherein the tocopherol comprises
.alpha.-tocopherol.
22. A method for making a paclitaxel/tocopherol-containing
emulsion, comprising: a) combining paclitaxel and polyethylene
glycol to provide a first paclitaxel-containing solution; b) adding
a tocopherol/polyethylene glycol conjugate to the first
paclitaxel-containing solution to provide a second
paclitaxel-containing solution; c) adding a tocopherol to the
second paclitaxel-containing solution to provide a third
paclitaxel-containing solution, d) blending the third
paclitaxel-containing solution with an aqueous phase to form a
pre-emulsion; and e) homogenizing the pre-emulsion to form an
emulsion.
23. The method of claim 22, wherein the polyethylene glycol has a
molecular weight between 200 and 600.
24. The method of claim 22, wherein the tocopherol/polyethylene
glycol conjugate comprises d-.alpha.-tocopherol polyethylene glycol
1000 succinate.
25. The method of claim 22, wherein the tocopherol comprises
.alpha.-tocopherol.
26. The method of claim 18 further comprising adding a
polyoxypropylene-polyoxyethylene glycol nonionic block polymer to
the first, second, or third paclitaxel-containing solution.
27. The method of claim 22 further comprising adding a
polyoxypropylene-polyoxyethylene glycol nonionic block polymer to
the first, second, or third paclitaxel-containing solution.
28. A method for making a paclitaxel-containing solution,
comprising: a) combining paclitaxel and polyethylene glycol to
provide a first paclitaxel solution; and b) adding a tocopherol
polyethylene glycol a polyoxypropylene-polyoxyethylene glycol
nonionic block polymer and a tocopherol to the first paclitaxel
solution to provide a second paclitaxel solution.
29. A method for making a paclitaxel-containing emulsion,
comprising: a) combining paclitaxel and polyethylene glycol to
provide a first paclitaxel solution; b) adding a tocopherol
polyethylene glycol a polyoxypropylene-polyoxyethylene glycol
nonionic block polymer and a tocopherol to the first paclitaxel
solution to provide a second paclitaxel solution; and c) blending
the second paclitaxel solution with an aqueous phase to provide an
emulsion.
30. The method of claim 29 further comprising homogenizing the
emulsion.
Description
FIELD OF THE INVENTION
This invention is in the field of pharmaceutical agents. In
particular, this invention relates to pharmaceutical agents wherein
tocopherol is used as a primary solvent.
BACKGROUND OF THE INVENTION
Hundreds of medically useful compounds are discovered each year,
but clinical use of these drugs is possible only if a drug delivery
vehicle is developed to transport them to their therapeutic target
in the human body. This problem is particularly critical for drugs
requiring intravenous injection in order to reach their therapeutic
target or dosage but which are water insoluble or poorly water
soluble. For such hydrophobic compounds, direct injection may be
impossible or highly dangerous, and can result in hemolysis,
phlebitis, hypersensitivity, organ failure and/or death. Such
compounds are termed by pharmacists "lipophilic", "hydrophobic", or
in their most difficult form, "amphiphobic".
A few examples of therapeutic substances in these categories are
ibuprofen, diazepam, griseofulvin, cyclosporin, cortisone,
proleukin, etoposide and paclitaxel. Kagkadis, K A et al. (1996)
PDA J Pharm Sci Tech 50(5):317 323; Dardel, O. 1976. Anaesth Scand
20:221 24. Sweetana, S and M J U Akers. (1996) PDA J Pharm Sci Tech
50(5):330 342.
Administration of chemotherapeutic or anti-cancer agents is
particularly problematic. The majority of these agents are poorly
soluble and thus are difficult to deliver in aqueous solvents and
supply at therapeutically useful levels. On the other hand,
water-soluble anti-cancer agents are generally taken up by both
cancer and non-cancer cells, thereby exhibiting
non-specificity.
Efforts to improve water-solubility and comfort of administration
of such agents have not solved, and may have worsened, the two
fundamental problems of cancer chemotherapy: 1) non-specific
toxicity and 2) rapid clearance form the bloodstream by
non-specific mechanisms. In the case of cytotoxins, which form the
majority of currently available chemotherapies, these two problems
are clearly related. Whenever the therapeutic is taken up by
non-cancerous cells, a diminished amount of the drug remains
available to treat the cancer, and more importantly, the normal
cell ingesting the drug is killed.
To be effective in treating cancer, the chemotherapeutic must be
present throughout the affected tissue(s) at high concentration for
a sustained period of time so that it may be taken up by the cancer
cells, but not at so high a concentration that normal cells are
injured beyond repair. Obviously, water soluble molecules can be
administered in this way, but only by slow, continuous infusion and
monitoring, aspects which entail great difficulty, expense and
inconvenience.
A more effective method of administering a cancer therapeutic,
particularly a cytotoxin, is in the form of a dispersion of oil in
which the drug is dissolved. These oily particles are made
electrically neutral and coated in such a way that they do not
interact with plasma proteins and are not trapped by the
reticuloendothelial system (RES), instead remaining intact in the
tissue or blood for hours, days or even weeks. It is desirable when
the particles also distribute themselves into the surrounding lymph
nodes which are injected at the site of a cancer. Nakamoto, Y et
al. (1975) Chem Pharm Bull 23(10):2232 2238. Takahashi, T et al.
(1977) Tohoku J Exp Med 123:235 246. In many cases direct injection
into blood is the route of choice for administration. Even more
preferable, following intravenous injection, the blood-borne
particles may be preferentially captured and ingested by the cancer
cells themselves. An added advantage of a particulate emulsion for
the delivery of a chemotherapeutic is the widespread property of
surfactants used in emulsions to overcome multidrug resistance.
For drugs that cannot be formulated as an aqueous solution,
emulsions have typically been most cost-effective and gentle to
administer, although there have been serious problems with making
them sterile and endotoxin free so that they may be administered by
intravenous injection. The oils typically used for pharmaceutical
emulsions include saponifiable oils from the family of
triglycerides, for example, soybean oil, sesame seed oil,
cottonseed oil, safflower oil and the like. Hansrani, P K et al.,
(1983) J. Parenter Sci. Technol 37:145 150. One or more surfactants
are used to stabilize the emulsion, and excipients are added to
render the emulsion more biocompatible, stable and less toxic.
Lecithin from egg yolks or soybeans is a commonly used surfactant.
Sterile manufacturing can be accomplished by absolute sterilization
of all the components before manufacture, followed by absolutely
aseptic technique in all stages of manufacture. However, improved
ease of manufacture and assurance of sterility is obtained by
terminal sterilization following sanitary manufacture, either by
heat or by filtration. Unfortunately, not all emulsions are
suitable for heat or filtration treatments.
Stability has been shown to be influenced by the size and
homogeneity of the emulsion. The preferred emulsion consists of a
suspension of sub-micron particles, with a mean droplet diameter of
no greater than 200 nanometers. A stable dispersion in this size
range is not easily achieved, but has the benefit that it is
expected to circulate longer in the bloodstream. Further, less of
the stable dispersion in this size range is phagocytized
non-specifically by the reticuloendothelial system. As a result the
drug is more likely to reach its therapeutic target. Thus, a
preferred drug emulsion will be designed to be actively taken up by
the target cell or organ, and is targeted away from the RES.
The use of vitamin E in emulsions is known. In addition to the
hundreds of examples where vitamin E in small quantities (for
example, less than 1%, Lyons, R. T., Pharm Res 13(9): S-226, (1996)
"Formulation development of an injectable oil-in-water emulsion
containing the lipophilic antioxidants .alpha.-tocopherol and
.beta.-carotene") is used as an anti-oxidant in emulsions, the
first primitive, injectable vitamin E emulsions per se were made by
Hidiroglou for dietary supplementation in sheep and for research on
the pharmacokinetics of vitamin E and its derivatives. Hidiroglou
M. and Karpinski K. (1988) Brit J Nutrit 59:509 518.
For mice, an injectable form of vitamin E was prepared by Kato and
coworkers. Kato Y., et al. (1993) Chem Pharm Bull 41(3):599 604.
Micellar solutions were formulated with Tween 80, Brij 58 and
HCO-60. Isopropanol was used as a co-solvent, and was then removed
by vacuum evaporation; the residual oil glass was then taken up in
water with vortexing as a micellar suspension. An emulsion was also
prepared by dissolving vitamin E with soy phosphatidycholine
(lecithin) and soybean oil. Water was added and the emulsion
prepared with sonication.
In 1983, E-Ferol, a vitamin B emulsions was introduced for vitamin
E supplementation and therapy in neonates. Alade, S. L., et al.
(1986) Pediatrics 77(4):593 597. Within a few months over 30 babies
had died as a result of receiving the product, and the product was
promptly withdrawn by FDA order. The surfactant mixture used in
E-Ferol to emulsify 25 mg/mL vitamin E consisted of 9% Tween 80 and
1% Tween 20. These surfactants at the employed levels seem
ultimately to have been responsible for the unfortunate deaths.
This experience illustrates the need for improved formulations and
the importance of selecting suitable biocompatible surfactants and
carefully monitoring their levels in parenteral emulsions.
An alternative means of solubilizing low solubility compounds is
direct solubilization in a non-aqueous milieu, for examples alcohol
(such as ethanol) dimethylsulfoxide or triacetin. An example in PCT
application WO 95/11039 describes the use of vitamin E and the
vitamin E derivative TPGS in combination with ethanol and the
immuno-suppressant molecule cyclosporin. U.S. Pat. No. 5,689,846
discloses various alcohol solutions of paclitaxel. U.S. Pat. No.
5,573,781 discloses the dissolution of paclitaxel in ethanol,
butanol and hexanol and an increase in the antitumor activity of
paclitaxel when delivered in butanol and hexanol as compared to
ethanol. Alcohol-containing solutions can be administered with
care, but are typically given by intravenous drip to avoid the
pain, vascular irritation and toxicity associated with bolus
injection of these solutions.
PCT publication WO 95/21217 (Dumex Ltd) discloses that tocopherols
can be used as solvents and/or emulsifiers of drugs that are
substantially insoluble in water, in particular for the preparation
of topical formulations. The use of vitamin E-TPGS as an emulsifier
in formulations containing high levels of .alpha.-tocopherol is
mentioned in the specification (pages 7 8 and 12). Examples 1 to 5
disclose formulations for topical administration comprising a lipid
layer (.alpha.-tocopherol), the drug and vitamin E-TPGS, in
quantities of less than 25% w/w of the formulation, as an
emulsifier. WO95/2 1217 does not suggest or describe anticancer
agents or taxanes.
PCT Publication WO 97/03651 (Danbiosyst UK Ltd.) discloses lipid
vehicle drug delivery compositions that contain at least five
ingredients: a therapeutic drug, vitamin E, an oil in which the
drug and vitamin E are dissolved, a stabilizer (either
phospholipid, a lecithin, or a poloxamer which is a
polyoxyethylene-polyoxypropylene copolymer) and water. The
therapeutic drugs disclosed are itraconazole and paclitaxel. The
"therapeutic emulsion" compositions require two oils in the
dispersed phase where the therapeutic drug resides, vitamin E and
another oil, typically a triglyceride such as soybean oil. The only
working example with paclitaxel, Example 16, also contains both
vitamin E and soybean oil.
N-methyl-2-pyrrolidone (NMP), under the trade name Pharmosolve.TM.,
can be used to improve the solubility of poorly soluble drugs in
pharmaceutical formulations and has appeared in recent literature
for use in veterinary medicine with forthcoming application in
humans. Furthermore, polyvinylpyrrolidone (PVP) under the trade
name Povidone.TM. with a molecular weight between 2,500 to 100,000
at a concentration of 1 to 5 percent (w/v) of the aqueous
injectable base can be used as a co-solubilizer along with NMP.
U.S. Pat. No. 5,726,181 discloses antitumor compositions and
suspensions comprising NMP and highly lipophilic camptothecin
derivatives.
Polyethylene glycols (PEGs) and PVP are examples of two
water-soluble polymers frequently used to modify the solubility
behavior of drugs, including paclitaxel. Although the solubility of
paclitaxel in both solvents is relatively high, in dilute aqueous
solutions that are suitable for parenteral administration the
solubility of the drug is low and the potential for drug
precipitation upon dilution is high. In admixtures of PEG 400 and
water containing 50 100% PEG 400, the solubility of paclitaxel
varies from 0.2 to 175 mg/ml, respectively. Thus, paclitaxel
solubilities are quite low where larger amounts of water are used,
e.g., solubilities in 35% PEG 400 and 30% PVP in water are 0.03
mg/ml and .ltoreq.0.3 mg/ml, respectively. "Solubility of
Paclitaxel in Polyethylene Glycol 400/Water Mixtures" Straubinger,
R. M., Biopharmaceutics of Paclitaxel (Taxol): Formulation.
Activity and Pharmacokinetics (p. 244), in: Taxol: Science and
Applications, (M. Suffness, ed.), CRC Press, Boca Raton, 1995). The
use of PEG-400 is not limited to paclitaxel and can be applied to
other therapeutic agents which exhibit good solubility in
polyethylene glycols (for example, Etoposide). Derivative forms of
paclitaxel including polyethylene glycol derivatives are described
in U.S. Pat. No. 5,614,549.
In addition to poor solubility and the potential for drug
precipitation with pharmaceutical formulations in non-aqueous
solvents such as alcohol (ethanol, isopropanol, benzyl alcohol,
etc.) along with surfactants, another problem is the ability of
these solvents to extract toxic substances, for examples
plasticizers, from their containers. The current commercial
formulation for the anti-cancer drug paclitaxel, for example,
consists of a mixture of hydroxylated castor oil and ethanol, and
rapidly extracts plasticizers such as di-(2-ethylhexyl)-phthalate
from commonly used intravenous infusion tubings and bags. Adverse
reactions to the plasticizers have been reported, such as
respiratory distress, necessitating the use of special infusion
systems at extra expense and time. Waugh, W. N., et al. (1991) Am.
J. Hosp. Pharmacists 48:1520.
In light of these problems, it can be seen that the ideal emulsion
vehicle would be inexpensive, non-irritating or even nutritive and
palliative in itself, terminally sterilizable by either heat or
filtration, stable for at least 1 year under controlled storage
conditions, accommodate a wide variety of water insoluble and
poorly soluble drugs and be substantially ethanol-free. In addition
to those drugs which are lipophilic and dissolve in oils, also
needed is a vehicle which will stabilize, and carry in the form of
an emulsion, drugs which are poorly soluble in lipids and in
water.
SUMMARY OF THE INVENTION
In order to meet these needs, the present invention is directed to
pharmaceutical compositions including: tocopherol, with and without
an aqueous phase, a surfactant or mixtures of surfactants
incorporating a co-solvent and a therapeutic agent. The
compositions of the invention may be in the form of an emulsion,
micellar solution or a self-emulsifying drug delivery system. The
tocopherol molecule is preferably .alpha.-tocopherol. The
compositions of the invention are generally substantially free of
any monohydric alcohol.
The co-solvent may include water-soluble polymers, preferably
polyethylene glycols or polyvinylpyrrolidone with or without
N-methyl-2-pyrrolidone. Polyethylene glycols (PEGs) with a
molecular weight between 100 to 10,000 are the most preferred
co-solvent. Most preferred is PEG-400 in amounts greater than 1% by
weight of the formulation.
The pharmaceutical compositions can be stabilized by the addition
of various amphiphilic molecules, including anionic, nonionic,
cationic, and zwitterionic surfactants. Preferably, these molecules
are PEGylated surfactants and optimally PEGylated
.alpha.-tocopherol.
The amphiphilic molecules further include surfactants such as
ascorbyl-6 palmitate, stearylamine, sucrose fatty acid esters,
PEGylated phospholipids, various vitamin E derivatives and
fluorine-containing surfactants (such as the Zonyl brand series)
and a polyoxypropylene-polyoxyethylene glycol nonionic block
copolymer.
The therapeutic agent of the emulsion may be a chemotherapeutic
agents preferably a taxoid analog and most preferably,
paclitaxel.
The emulsions of the invention can comprise an aqueous medium when
in the form of an emulsion or micellar solution. This medium can
contain various additives to assist in stabilizing the emulsion or
in rendering the formulation biocompatible.
In one form, the invention is directed to a pharmaceutical
composition comprising .alpha.-tocopherol, a chemotherapeutic
selected from taxoids, taxins and taxanes, water and
D-.alpha.-tocopherol polyethyleneglycol 1000 succinate. In another
form, the invention is directed to a pharmaceutic composition
comprising .alpha.-tocopherol, a co-solvent, one or more
surfactants, an aqueous phase and a therapeutic agent wherein the
composition is in the form of an emulsion or micellar solution and
the solution is substantially free of any monohydric alcohol.
In a preferred format, the co-solvent may be polyethylene glycol,
N-methyl-2-pyrrolidone, polyvinyl-pyrrolidone or mixtures
thereof.
In a preferred format, the surfactant is an .alpha.-tocopherol
derivative and the polyethylene glycol has a molecular weight
between 100 to 10,000, most preferably from about 200 to about
1000.
In a preferred format the therapeutic agent is a chemotherapeutic
agent selected from taxoids, taxines and taxanes.
The pharmaceutical compositions of the invention are typically
formed by dissolving a therapeutic agent in the co-solvent to form
a therapeutic agent solution; .alpha.-tocopherol is then added
along with one or more surfactants to the therapeutic agent
solution to form an oil solution of the therapeutic agent in the
hydrophilic co-solvent. The oil solution is then blended with an
aqueous phase to form a pre-emulsion. For IV delivery the
pre-emulsion is further homogenized to form a fine emulsion. For
oral delivery, the oil solution of the therapeutic agent in the
co-solvent along with surfactants is typically encapsulated in a
gelatin capsule.
In a preferred form of the method of the invention the therapeutic
agent is dissolved in polyethylene glycol which allows the
avoidance of the use of monohydric alcohols as a solvent.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be better understood by reference to the
figures, in which:
FIG. 1A shows the particle size of a paclitaxel emulsion (QWA) at
7.degree. C. over time;
FIG. 1B shows the particle size of a paclitaxel emulsion (QWA) at
25.degree. C. over time;
FIG. 2 is an HPLC chromatogram showing the integrity of a
paclitaxel in an emulsion as described in Example 5;
FIG. 3A shows the paclitaxel concentration of a paclitaxel emulsion
(QWA) at 4.degree. C. over time;
FIG. 3B shows the paclitaxel concentration of a paclitaxel emulsion
(QWA) at 25.degree. C. over time; and
FIG. 4 shows the percentage of paclitaxel released over time from
three different emulsions. The symbol .circle-solid. represents the
percentage of paclitaxel released over time from an emulsion
commercially available from Bristol Myers Squibb. The symbol
.tangle-solidup. represents the percentage of paclitaxel released
over time from an emulsion of this invention (QWB) containing 7
mg/ml paclitaxel (QWA) as described in Example 6. The symbol
.diamond. represents the percentage of paclitaxel released over
time from an emulsion of this invention containing 6 mg/ml
paclitaxel as described in Example 7.
FIG. 5 shows the efficacy of a PEG-400/Vitamin E/paclitaxel
emulsion against B16 melanoma in nice.
DETAILED DESCRIPTION OF THE INVENTION
To ensure a complete understanding of the invention the following
definitions are provided:
Tocopherols: Tocopherols are a family of natural and synthetic
compounds, also known by the generic names tocols or vitamin E.
.alpha.-tocopherol is the most abundant and active form of this
class of compounds, and it has the following chemical structure
(Scheme I):
##STR00001##
Other members of this class include .alpha.-, .beta.-, .gamma.-,
and .delta.-tocotrienols, and .alpha.-tocopherol derivatives such
as tocopherol acetate, phosphate, succinate, nitotinate and
linoleate. In addition to their use as a primary solvent,
tocopherols and their derivatives are useful as therapeutic
agents.
Surfactants: Surface active group of amphiphilic molecules which
are manufactured by chemical processes or purified from natural
sources or processes. These can be anionic, cationic, nonionic, and
zwitterionic. Typical surfactants are described in Emulsions:
Theory and Practice, Paul Becher, Robert E. Krieger Publishing,
Malabar, Fla., 1965; Pharmaceutical Dosage Forms: Dispersed Systems
Vol. 1, Martin M. Rigear, Surfactants and U.S. Pat. No. 5,595,723
which is assigned to the assignee of this invention, Sonus
Pharmaceuticals. All of these references are hereby incorporated by
reference.
TPGS: TPGS or PEGylated vitamin E is a vitamin E derivative in
which polyethylene glycol subunits are attached by a succinic acid
diester at the ring hydroxyl of the vitamin E molecule. TPGS stands
for D-.alpha.-tocopherol polyethyleneglycol 1000 succinate
(MW=1513). TPGS is a non-ionic surfactant (HLB=16 18) with the
structure of Scheme II:
##STR00002##
Various chemical derivatives of vitamin E TPGS including ester and
ether linkages of various chemical moieties are included within the
definition of vitamin E TPGS.
Polyethylene glycol: Polyethylene glycol (PEG) is a hydrophilic,
polymerized form of ethylene glycol, consisting of repeating units
of the chemical structure--(CH.sub.2--CH.sub.2--O--). The general
formula for polyethylene glycol is HOCH.sub.2
(CH.sub.2OCH.sub.2).sub.n CH.sub.2OH or
H(OCH.sub.2CH.sub.2).sub.nOH. The molecular weight ranges from 200
to 10,000. Such various forms are described as PEG-200, PEG-400 and
the like.
N-Methyl-2-pyrrolidone: N-methyl-2-pyrrolidone (NMP) is an organic
molecule with the following chemical structure:
##STR00003##
A GMP grade of this compound is available under the name
Pharmasolve.TM. and is used to improve the solubility of poorly
soluble drugs in pharmaceutical formulations. The enhanced
solubility of certain drugs can be attributed to a complexing
action with the nitrogen and carbonyl reactive centers of the
molecule.
Polyvinyl pyrrolidone: Polyvinyl pyrrolidone (PVP) or Povidone is a
water-soluble polymer, consisting of repeating units of the
chemical structure:
##STR00004##
Its average MW can vary between 2500 and 3.times.10.sup.6; special
grades of pyrogen-free povidone are available for parenteral
administration. Concentrations up to 5% w/v can be used as the
co-solvent for poorly soluble drugs.
Poloxamers or Pluronics: are synthetic block copolymers of ethylene
oxide and propylene oxide having the general structure:
H(OCH.sub.2CH.sub.2).sub.a(OC.sub.3H.sub.6).sub.b(OCH.sub.2CH.sub.2).sub.-
aOH
The following variants based on the values of a and b are
commercially available from BASF Performance Chemicals (Parsippany,
N.J.) under the trade name Pluronic and which consist of the group
of surfactants designated by the CTFA name of Poloxamer 108, 188,
217, 237, 238, 288, 338, 407, 101, 105, 122, 123, 124, 181, 182,
183, 184, 212, 231, 282, 331, 401, 402, 185, 215, 234, 235, 284,
333, 334, 335, and 403. For the most commonly used poloxamers 124,
188, 237, 338 and 407 the values of a and b are 12/20, 79/28,
64/37, 141/44 and 101/56, respectively.
Solutol HS-15: is a polyethylene glycol 660 hydroxystearate
manufactured by BASF (Parsippany, N.J.). Apart from free
polyethylene glycol and its monoesters, diesters are also
detectable. According to the manufacturer, a typical lot of Solutol
HS-15 contains approximately 30% free polyethylene glycol and 70%
polyethylene glycol esters.
Other surfactants: Other surfactants useful in the invention
include ascorbyl-6 palmitate (Roche Vitamins, Nutley, N.J.),
stearylamine, and sucrose fatty acid esters (Mitsubishi Chemicals).
Custom surfactants include those compounds with polar water-loving
heads and hydrophobic tails, such as a vitamin E derivative
comprising a peptide bonded polyglutamate attached to the ring
hydroxyl and pegylated phytosterol. Other peptides may be bonded to
vitamin E as well. Also pegylated phospholipids are useful
surfactants. Examples of pegylated phospholipids include PEG 2000
or PEG 5000 analogs of phosphatidylethanolamine where the fatty
acyl chains contain C.sub.6 C.sub.24 fatty acids which can be
saturated, unsaturated, mixtures thereof.
Hydrophile-lipophile balance: An empirical formula used to index
surfactants. Its value varies from 1 45 and in the case of
non-ionic surfactants from about 1 20. In general for lipophilic
surfactants the HLB is less than 10 and for hydrophilic ones the
HLB is greater than 10.
Biocompatible: Capable of performing functions within or upon a
living organism in an acceptable manner, without undue toxicity or
physiological or pharmacological effects.
Substantially free of any monohydric alcohol: A composition having
a monohydric alcohol concentration less than about 1.0% (w/v)
monohydric alcohol. As used herein, the term "monohydric" alcohol
is an alcohol containing one hydroxyl group, such as but not
limited to ethanol, butanol, isopropanol. The term "polyhydric"
alcohol or "polyol" is an alcohol containing two or more hydroxyl
groups, such as but not limited to, ethylene glycol, propylene
glycol or polyethylene glycol (PEG). PEG is also referred to as
polyglycol with ethylene glycol as a polymerized unit. Other
suitable polyhydric alcohols for use herein include, but are not
limited to, ethylene glycol (2-OH groups), glycerol (3-OH groups),
sorbitol (6-OH groups) and mannitol (6-OH groups).
Emulsion: A colloidal dispersion of two immiscible liquids in the
form of droplets, whose diameter, in general, is between 0.1 and
3.0 microns1 and which is typically optically opaque, unless the
dispersed and continuous phases are refractive index matched. Such
systems possess a finite stability, generally defined by the
application or relevant reference system, which may be enhanced by
the addition of amphiphilic molecules or viscosity enhancers.
Microemulsion: A thermodynamically stable isotropically clear
dispersion of two immiscible liquids, such as oil and water,
stabilized by an interfacial film of surfactant molecules. The
microemulsion has a mean droplet diameter of less than 200 nm, in
general between 10 50 nm. In the absence of water, mixtures of
oil(s) and non-ionic surfactant(s) form clear and isotropic
solutions that are known as self-emulsifying drug delivery systems
(SEDDS) and have successfully been used to improve lipophilic drug
dissolution and oral absorption.
PEGylated: PEGylated or ethoxylated means polyethylene glycol
subunits attached to a given compound via a chemical linkage.
Aqueous Medium: A water-containing liquid which can contain
pharmaceutically acceptable additives such as acidifying,
alkalizing, buffering, chelating, complexing and solubilizing
agents, antioxidants and antimicrobial preservatives, humectants,
suspending and/or viscosity modifying agents, tonicity and wetting
or other biocompatible materials.
Therapeutic Agent: Any compound, natural or synthetic, which has a
biological activity, is soluble in the oil phase and has an
octanol-buffer partition coefficient (Log P) of at least 2 to
ensure that the therapeutic agent is preferentially dissolved in
the oil phase rather than the aqueous phase. This includes
peptides, non-peptides and nucleotides. Hydrophobic derivatives of
water soluble molecules such as lipid conjugates/prodrugs are
within the scope of therapeutic agents.
Chemotherapeutic: Any natural or synthetic molecule which is
effective against one or more forms of cancer, and particularly
those molecules which are slightly or completely lipophilic or
which can be modified to be lipophilic. This definition includes
molecules which by their mechanism of action are cytotoxic
(anti-cancer agents), those which stimulate the immune system
(immune stimulators) and modulators of angiogenesis. The outcome in
either case is the slowing of the growth of cancer cells.
Chemotherapeutics include Taxol (paclitaxel) and related molecules
collectively termed taxoids, taxines or taxanes. The structure of
paclitaxel is shown in the figure below (Scheme V).
##STR00005##
Included within the definition of "taxoids" are various
modifications and attachments to the basic ring structure (taxoid
nucleus) as may be shown to be efficacious for reducing cancer cell
growth and to partition into the oil (lipid phase) and which can be
constructed by organic chemical techniques known to those skilled
in the art. These include but are not limited to benzoate
derivatives of paclitaxel, such as 2-debenzoyl-2-aroyl and
C-2-acetoxy-C-4-benzoate paclitaxel, 7-deocytaxol, C-4 aziridine
paclitaxel, as well as various paclitaxel conjugates with natural
and synthetic polymers, particularly with fatty acids,
phospholipids, and glycerides and 1,2-diacyloxypropane-3-amine.
Docetaxel (Taxotere) is also a preferred taxane. The structure of
the taxoid nucleus is shown in Scheme VI.
##STR00006##
Also included within the scope of the present invention are natural
products that share structural similarities with paclitaxel, i.e.,
they incorporate a common pharmacophore proposed for
microtubule-stabilizing agents. These compounds include but are not
limited to epothilone A and B, discodermolide, nonataxel and
eleutherobin (Chem. Eng. News (1999) 77(17):35 36).
Chemotherapeutics include podophyllotoxins and their derivatives
and analogues. The core ring structure of these molecules is shown
in the following figure (Scheme VII):
##STR00007##
Another important class of chemotherapeutics useful in this
invention are camptothecins, the basic ring structure of which is
shown in the following figure, but includes any derivatives and
modifications to this basic structure which retain efficacy and
preserve the lipophilic character of the molecule shown below
(Scheme VIII).
##STR00008##
Another preferred class of chemotherapeutics useful in this
invention are the lipophilic anthracyclines, the basic ring
structure of which is shown in the following figure (Scheme
IX):
##STR00009##
Suitable lipophilic modifications of Scheme IX include
substitutions at the ring hydroxyl group or sugar amino group.
Another important class of chemotherapeutics are compounds which
are lipophilic or can be made lipophilic by molecular
chemosynthetic modifications well known to those skilled in the
art, for example by combinatorial chemistry and by molecular
modelling, and are drawn from the following list: Taxotere,
Amonafide, Illudin S, 6-hydroxymethylacylfulvene Bryostatin 1,
26-succinylbryostatin 1, Palmitoyl Rhizoxin, DUP 941, Mitomycin B,
Mitomycin C, Penclomedine. Interferon .alpha.2b, angiogenesis
inhibitor compounds, Cisplatin hydrophobic complexes such as
2-hydrazino-4,5-dihydro-1H-imidazole with platinum chloride and
5-hydrazino-3,4-dihydro-2H-pyrrole with platinum chloride, vitamin
A, vitamin E and its derivatives, particularly tocopherol
succinate.
Other compounds useful in the invention include:
1,3-bis(2-chloroethyl)-1-nitrosurea ("carmustine" or "BCNU"),
5-fluorouracil, doxorubicin ("adriamycin"), epirubicin,
aclarubicin, Bisantrene
(bis(2-imidazolen-2-ylhydrazone)-9,10-anthracenedicarboxaldehyde,
mitoxantrone, methotrexate, edatrexate, muramyl tripeptide, muramyl
dipeptide, lipopolysaccharides, 9-b-d-arabinofuranosyladenine
("vidarabine") and its 2-fluoro derivative, resveratrol, retinoic
acid and retinol, Carotenoids, and tamoxifen.
Other compounds useful in the application of this invention
include: Decarbazine, Lonidamine, Piroxantrone, Anthrapyrazoles,
Etoposide, Camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin,
camptothecin-11 ("Irinotecan"), Topotecan, Bleomycin, the Vinca
alkaloids and their analogs [Vincristine, Vinorelbine, Vindesine,
Vintripol, Vinxaltine, Ancitabine], 6-aminochrysene, and
navelbine.
Other compounds useful in the application of the invention are
mimetics of taxol, eleutherobins, sarcodictyins, discodermolides
and epothiolones.
Other compounds useful in the invention are microtubule targeting
agents. Microtubule targeting agents may bind to a protein called
tubulin and thus prevent microtubule polymerization. Representative
microtubule binding agents include epothilones, elutherobin and
discodermolide.
Taking into account these definitions, the present invention is
directed to pharmaceutical compositions in the form of emulsions,
micellar solutions or self-emulsifying drug delivery systems which
are substantially free of ethanol solvent.
The therapeutic agents of the compositions of this invention can
initially be solubilized in a co-solvent. In the case of ethanol
during the preparation of the oil phases the ethanol is removed and
a substantially ethanol-free composition is formed. The ethanol
concentration is less than 1% (w/v), preferably less than 0.5%, and
most preferably less than 0.3%. The therapeutic agents can also be
solubilized in methanol, propanol, chloroform, isopropanol, butanol
and pentanol. These solvents are also removed prior to use.
In a preferred embodiment, the therapeutic agents of the
compositions of the invention can initially be solubilized in
non-volatile co-solvents such as dimethylsulfoxide (DMSO),
dimethylamide (DMA), propylene glycol (PG), polyethylene glycol
(PEG), N-methyl-2-pyrrolidone (NMP) and polyvinylpyrrolidone (PVP);
NMP or a water-soluble polymer such as PEG or PVP (Table 1) are
particularly preferred.
A major advantage/improvement of using PEG-400 to solubilize
therapeutic agents rather than alcohols such as ethanol is that a
volatile solvent does not have to be removed or diluted prior to
administration of the therapeutic agent. The final polyethylene
glycol levels in the emulsion can be varied from 1 50%, preferably
from 1 25% and more preferably from 1 10% (w/w). Suitable
polyethylene glycol solvents are those with an average molecular
weight between 200 and 600, preferably between 300 and 400 (Table
1). In the case of self-emulsified systems for oral administration,
high molecular weight PEGs (1,000 10,000) can also be included as
solidification agents to form semi-solid formulations which can be
filled into hard gelatin capsules.
TABLE-US-00001 TABLE 1 Physical Properties of Low Molecular Weight
Polyethylene Glycols Physical Property PEG 200 PEG 300 PEG 400 PEG
600 Molecular Weight 190 210 285 315 380 420 570 630 Viscosity
(mPas) 46 53 66 74 85 95 130 150 Refractive Index 1.459 1.463 1.465
1.467 (25.degree. C.) Freezing point (.degree. C.) -50 -16 to -12
-3 to 8 15 to 25
Solubilization of the therapeutic agents of the invention in
polyethylene glycol or other non-volatile co-solvents (PVP, NMP)
avoids the necessity of solubilizing the therapeutic agents of the
invention in monohydric alcohols such as ethanol or other volatile
solvents. Use of polyethylene glycol or N-methyl-2-pyrrolidone
eliminates the need to remove the solvent prior to use of the
emulsions therapeutically.
The final polyethylene glycol levels in the emulsion can be varied
from 1 50%, preferably from 1 25% and more preferably from 1 10%
(w/w).
The compositions of the invention contain tocopherol as a carrier
for therapeutic drugs, which can be administered to animals or
humans via intravascular, oral, intramuscular, cutaneous and
subcutaneous routes. Specifically, the emulsions can be given by
any of the following routes, among others: intraabdominal,
intraarterial, intraarticular, intracapsular, intracervical,
intracranial, intraductal, intradural, intralesional, intralocular,
intralumbar, intramural, intraocular, intraoperative,
intraparietal, intraperitoneal, intrapleural, intrapulmonary,
intraspinal, intrathoracic, intratracheal, intratympanic,
intrauterine, and intraventricular. The emulsions of the present
invention can be nebulized using suitable aerosol propellants which
are known in the art for pulmonary delivery of lipophilic
compounds.
In its first aspect, the invention is directed to the use of
tocopherol as the hydrophobic dispersed phase of emulsions
containing water insoluble, poorly water soluble therapeutic
agents, water soluble ones which have been modified to be less
water soluble or mixtures thereof. In a preferred embodiment
.alpha.-tocopherol is employed. Also called vitamin E,
.alpha.-tocopherol is not a typical lipid oil. It has a higher
polarity than most lipid oils, particularly triglycerides, and is
not saponifiable. It has practically no solubility in water.
In the second aspect, the invention is an .alpha.-tocopherol
emulsion in the form of a self-emulsifying system where the system
is to be used for the oral administration of water-insoluble (or
poorly water-soluble or water-soluble agents modified to be less
water soluble or mixtures thereof) drugs where that is desired. In
this embodiment, an oil phase with surfactant and drug or drug
mixture is encapsulated into a soft or hard gelatin capsule.
Suitable solidification agents with melting points in the range of
40 to 60.degree. C., such as high molecular weight polyethylene
glycols (MW>1000), and glycerides, such as those available under
the trade name Gelucire (Gattefose Corp., Saint Priest, France),
can be added to allow filling of the formulation into a hard
gelatin capsule at a high temperature. Semi-solid formulations are
formed upon room temperature equilibration. Upon dissolution of the
gelatin in the stomach and duodenum, the oil is released and forms
a fine emulsion with a mean droplet diameter of between 2 5 microns
spontaneously. The emulsion is then taken up by the microvilli of
the intestine and released into the bloodstream.
In a third aspect, the invention comprises microemulsions
containing tocopherol, preferably .alpha.-tocopherol.
Microemulsions refer to a sub-class of emulsions where the emulsion
suspension is essentially clear and indefinitely stable by virtue
of the extremely small size of the oil/drug microaggregates
dispersed therein.
In a fourth aspect of the invention, PEGylated vitamin E (TPGS) is
used as a primary surfactant in emulsions of vitamin E. PEGylated
vitamin E is utilized as a primary surfactant, a stabilizer and
also as a supplementary solvent in emulsions of vitamin E.
Polyethylene glycol (PEG) is also useful as a co-solvent in the
emulsions of this invention. Of particular use is polyethylene
glycol 200, 300, 400 or mixtures thereof.
The .alpha.-tocopherol concentration of the emulsions of this
invention can be from about 1 to about 10% w/v. The ratio of
.alpha.-tocopherol to TPGS is optimally from about 1:1 to about
10:1 (w/w).
The emulsions of the invention may further include surfactants such
as ascorbyl-6 palmitate, stearylamine, PEGylated phospholipids,
sucrose fatty acid esters and various vitamin E derivatives
comprising Q-tocopherol nicotinate, tocopherol phosphate, and
nonionic, synthetic surfactant mixtures, such as
polyoxypropylene-polyoxyethylene glycol nonionic block
copolymer.
The emulsions of the invention can comprise an aqueous medium. The
aqueous phase generally has an osmolality of approximately 300 mOsm
and may include sodium chloride, sorbitol, mannitol, polyethylene
glycol, propylene glycol albumin, polypep and mixtures thereof.
This medium can also contain various additives to assist in
stabilizing the emulsion or in rendering the formulation
biocompatible. Acceptable additives include acidifying agents,
alkalizing agents, antimicrobial preservatives, antioxidants,
buffering agents, chelating agents, suspending and/or
viscosity-increasing agents, and tonicity agents. Preferably,
agents to control the pH, tonicity, and increase viscosity are
included. Optimally, a tonicity of at least 250 mOsm is achieved
with an agent which also increases viscosity, such as sorbitol or
sucrose.
The emulsions of the invention for intravenous injection have a
particle size (mean droplet diameter) of 10 to 500 nm, preferably
10 to 200 nm and most preferably 10 to 100 nm. For intravenous
emulsions, the spleen and liver will eliminate particles greater
than 500 nm in size through the RES.
A preferred form of the invention includes paclitaxel, a very
water-insoluble cytotoxin used in the treatment of uterine cancer
and other carcinomas. An emulsion composition of the present
invention comprises a solution of vitamin E containing paclitaxel
at a concentration of up to 20 mg/mL, four times that currently
available by prescription, and a biocompatible surfactant such that
the emulsion microdroplets are less than 0.2 microns and are
terminally sterilizable by filtration.
Preferred injectable compositions contain: 0.1 1.0% paclitaxel (1
10 mg/ml); 1 10% PEG400; 3 10% Vitamin E; 1 6% TPGS and 0.5 2.5%
Pluronic F127.
Another preferred composition contains: 1.0 paclitaxel (10 mg/ml),
6% PEG400, 8% Vitamin E, 5% TPGS, 1% Pluronic F127 and 80% aqueous
solution.
Preferred formulations for self-emulsifying systems are as follows:
0.1 20% paclitaxel, 10 90% Vitamin E, 10 90% PEG 400 or
N-methyl-2-pyrrolidone, 5 50% TPGS, 5 50% a secondary hydrophilific
surfactant, such as Polysorbates (Tween 80), Pluronics (Pluronic
F127) or Cremophor EL/RH40, Solutol HS-15). The oil phase (vitamin
E) can optionally contain polyvinylpyrrolidone, glycerol and
propylene glycol esters such as mono-/di-/triglycerides and
mono-diesters of propylene glycol. In addition, high MW PEGs (1,000
10,000) and high melting point glycerol esters can be included to
provide the formulation with semisolid consistency.
A further embodiment of the invention is a method of treating
carcinomas comprising the parenteral administration of a bolus dose
of paclitaxel in vitamin E emulsion with and without PEGylated
vitamin E by intravenous injection once daily or every second day
over a therapeutic course of several weeks. Such method can be used
for the treatment of carcinomas of the breast, lung, skin and
uterus.
The general principles of the present invention may be more fully
appreciated by reference to the following non-limiting
examples.
EXAMPLES
Example 1
Dissolution of Paclitaxel in .alpha.-Tocopherol
.alpha.-tocopherol was obtained from Sigma Chemical Company (St.
Louis, Mo.) in the form of a synthetic dl-.alpha.-tocopherol of 95%
purity prepared from phytol. The oil was amber in color and very
viscous. Paclitaxel was purchased from Hauser Chemical Research
(Boulders Colo.) and was 99.9% pure by HPLC. Paclitaxel 200 mg was
dissolved in 6 mL of dry absolute ethanol (Spectrum Chemical
Manufacturing Corn, Gardena, Calif.) and added to 1 gm
.alpha.-tocopherol. The ethanol was then removed by vacuum at
42.degree. C. until the residue was brought to constant weight.
Independent studies showed that the ethanol content was less than
0.3% (w/v).
The resultant solution was clear, amber and very viscous, with a
nominal concentration of 200 mg/gm (w/w) paclitaxel in
.alpha.-tocopherol. Higher concentrations of paclitaxel (up to 400
mg/gm, w/w) can be solubilized in .alpha.-tocopherol.
Example 2
Anionic Surfactant Used to Prepare .alpha.-tocopherol Emulsions
Paclitaxel 2 gm in 10 gm of .alpha.-tocopherol, prepared as
described in Example 1, was emulsified with ascorbyl palmitate as
the triethanolamine salt by the following method. A solution
consisting of ascorbic acid 20 mM was buffered to pH 6.8 with
triethanolamine as the free base to form 2.times. buffer. 50 mL of
the 2.times. buffer was placed in a Waring blender. 0.5 gm of
ascorbyl-6-palmitate (Roche Vitamins and Fine Chemicals, Nutley,
N.J.), an anionic surfactant, was added and the solution blended at
high speed for 2 min at 40.degree. C. under argon. The
.alpha.-tocopherol containing paclitaxel was then added into the
blender with the surfactant and buffer. Mixing was continued under
argon until a coarse, milky, pre-emulsion was obtained,
approximately after 1 min at 40.degree. C. Water for injection was
then added, bringing the final volume to 100 mL.
The pre-emulsion was transferred to the feed vessel of a
Microfluidizer Model 110Y (Microfluidics Inc, Newton, Mass.). The
unit was immersed in a bath to maintain a process temperature of
approximately 60.degree. C. during homogenization, and was flushed
with argon before use. After priming, the emulsion was passed
through the homogenizer in continuous re-cycle for 10 minutes at a
pressure gradient of about 18 kpsi across the interaction head. The
flow rate was about 300 mL/min, indicating that about 25 passes
through the homogenizer resulted.
The resultant paclitaxel emulsion in an .alpha.-tocopherol vehicle
was bottled in amber vials under argon and stored with
refrigeration at 7.degree. C. and 25.degree. C. Samples were taken
at discrete time intervals for particle sizing and chemical
analysis.
Data taken with a Nicomp Model 370 Submicron Particle Sizer
(Particle Sizing Systems Inc, Santa Barbara, Calif.) showed that
the emulsion had a mean particle diameter of 280 nm.
Example 3
Use of PEGylated Vitamin E (TPGS)
A ternary phase diagram was constructed for .alpha.-tocopherol,
PEGylated vitamin E (TPGS, vitamin-E
polyoxyethyleneglycol-1000-succinate, obtained from Eastman
Chemical Co., Kingsport, Tenn.), and water. TPGS was first melted
at 42.degree. C. and mixed gravimetrically with .alpha.-tocopherol
at various proportions from 1 to 100% TPGS, the balance being
.alpha.-tocopherol. Mixtures were miscible at all concentrations.
Water was then added to each mixture in such a way that the final
water concentration was increased stepwise from zero to 97.5%. At
each step, observations were made of the phase behavior of the
mixture. As appropriate, mixing was performed by vortexing and
sonication, and the mixture was heated or centrifuged to assess its
phase composition.
A broad area of biphasic o/w emulsions suitable for parenteral
administration was found at water concentrations above 80%. The
emulsions formed were milky white, free flowing liquids that
contained disperse .alpha.-tocopherol microparticles stabilized by
non-ionic surfactant. Also in this area, microemulsions potentially
suitable as drug carriers were observed at TPGS to oil ratios above
about 1:1. At lower water content, a broad area containing
transparent gels (reverse emulsions) was noted. Separating the two
areas (high and low water content) is an area composed of opaque,
soap-like liquid crystals.
Phase diagrams of .alpha.-tocopherol with surfactant combinations,
for example TPGS with a nonionic, anionic or cationic co-surfactant
(for example glutamyl stearate, ascorbyl palmitate or Pluronic
F-68), or drug can be prepared in a similar manner.
Example 4
.alpha.-Tocopherol Emulsion for Intravenous Delivery of
Paclitaxel
A formulation of the following composition was prepared:
TABLE-US-00002 paclitaxel 1.0 gm % .alpha.-tocopherol 3.0 gm % TPGS
2.0 gm % Ascorbyl-6-Palmitate 0.25 gm % Sorbitol 5.0 gm %
Triethanolamine to pH 6.8 Water qs to 100 mL
The method of preparation was as follows: synthetic
.alpha.-tocopherol (Roche Vitamins, Nutley, N.J.), paclitaxel
(Hauser, Boulder, Colo.), ascorbyl 6-palmitate (Aldrich Chemical
Co, Milwaukee, Wis.) and TPGS were dissolved in 10 volumes of
anhydrous undenatured, ethanol (Spectrum Quality Products,
Gardenia, Calif.) with heating to 40 45.degree. C. The ethanol was
then drawn off with vacuum until no more than 0.3% remained by
weight.
Pre-warmed aqueous solution containing a biocompatible osmolyte and
buffer were added with gentle mixing, and a white milk formed
immediately. This mixture was further improved by gentle rotation
for 10 minutes with continuous warming at 40 45.degree. C. This
pre-mixture at about pH 7 was then further emulsified as described
below.
The pre-mixture at 40 45.degree. C. was homogenized in an Avestin
C5 homogenizer (Avestin, Ottawa Canada) at 26 Kpsi for 12 minutes
at 44.degree. C. The resultant mixture contained microparticles of
.alpha.-tocopherol with a mean size of about 200 nm. Further pH
adjustment was made with an alkaline 1 M solution of
triethanolamine (Spectrum Quality Products).
In order to avoid gelation of the TPGS during the early stage of
emulsification, all operations were performed above 40.degree. C.
and care was taken to avoid exposure of the solutions to cold air
by covering all vessels containing the mixture. Secondly, less than
2% TPGS should generally be dissolved in .alpha.-tocopherol oil
before pre-emulsification, the balance of the TPGS being first
dissolved in the aqueous buffer before the pre-emulsion is
prepared. The solution gels at concentrations of TPGS higher than
2%.
Physical stability of the emulsion was then examined by placing
multiple vials on storage at 4.degree. C. and 25.degree. C. Over
several months, vials were periodically withdrawn for particle
sizing. Mean particle size, as determined with the Nicomp Model 370
(Particle Sizing Systems, Santa Barbara, Calif.), is shown for the
two storage temperatures in FIG. 1. The particle size distribution
was bi-modal.
Example 5
Chemical Stability of Paclitaxel in an .alpha.-Tocopherol
Emulsion
After emulsification, the formulation of Example 4 was analyzed for
paclitaxel on a Phenosphere CN column (5 microns, 150.times.4.6
mm). The mobile phase consisted of a methanol/water gradient, with
a flow rate of 1.0 mL/min. A UV detector set at 230 nm was used to
detect and quantitate paclitaxel. A single peak was detected (FIG.
2), which had a retention time and mass spectrogram consistent with
native reference paclitaxel obtained from Hauser Chemical (Boulder,
Colo.).
Chemical stability of the emulsion of Example 4 was examined by
HPLC during storage. The data of FIG. 3 demonstrate that paclitaxel
remains stable in the emulsion for periods of at least 3 months,
independent of the storage temperature. Taken together, the data of
FIGS. 2 and 3 demonstrate successful retention of drug potency and
emulsion stability when stored at 4.degree. C. for a period of at
least 3 months.
Example 6
Paclitaxel Emulsion Formulation QWA
An emulsion of paclitaxel 10 mg/ml for intravenous drug delivery,
having the following composition, was prepared as described in
Example 4.
TABLE-US-00003 paclitaxel 1.0 gm % .alpha.-tocopherol 3.0 gm % TPGS
1.5 gm % Ascorbyl-6-Palmitate 0.25 gm % Sorbitol 4.0 gm %
Triethanolamine to pH 6.8 Water qs to 100 mL
Example 7
Paclitaxel Emulsion Formulation QWB
A second emulsion of paclitaxel 10 mg/ml for intravenous drug
delivery, having the following composition, was prepared as
described in Example 4.
TABLE-US-00004 paclitaxel 1.0 gm % .alpha.-tocopherol 3.0 gm % TPGS
1.5 gm % Solutol HS-15 1.0 gm % Sorbitol 4.0 gm % Triethanolamine
to pH 6.8 Water qs to 100 mL Solutol HS-15 is a product of BASF
Corp, Mount Olive NJ.
Example 8
10 mg/mL Paclitaxel Emulsion Formulation QWC
A third emulsion formulation of paclitaxel 10 mg/ml was prepared as
follows using Poloxamer 407 (BASF Corp, Parsippany, N.J.) as a
co-surfactant.
TABLE-US-00005 paclitaxel 1.0 gm % .alpha.-tocopherol 6.0 gm % TPGS
3.0 gm % Poloxamer 407 1.0 gm % Sorbitol 4.0 gm % Triethanolamine
to pH 6.8 Water for injection qs to 100 mL
In this example, 1.0 gm Poloxamer 407 and 1.0 gm paclitaxel were
dissolved in 6.0 gm .alpha.-tocopherol with ethanol 10 volumes and
gentle heating. The ethanol was then removed under vacuum.
Separately, an aqueous buffer was prepared by dissolving 3.0 gm
TPGS and 4.0 gm sorbitol in a final volume of 90 mL water for
injection. Both oil and water solutions were warmed to 45.degree.
C. and mixed with sonication to make a pre-emulsion. A vacuum was
used to remove excess air from the pre-emulsion before
homogenization.
Homogenization was performed in an Avestin C5 as already described.
The pressure differential across the homogenization valve was 25
kpsi and the temperature of the feed was 42.degree. 45.degree. C. A
chiller was used to ensure that the product exiting the homogenizer
did not exceed a temperature of 50.degree. C. Flow rates of 50
mL/min were obtained during homogenization. After about 20 passes
in a recycling mode, the emulsion became more translucent.
Homogenization was continued for 20 min. Samples were collected and
sealed in vials as described before. A fine .alpha.-tocopherol
emulsion for intravenous delivery of paclitaxel was obtained. The
mean particle diameter of the emulsion was 77 nm. Following
0.22.mu. sterile filtration through a 0.22 micron Durapore filter
(Millipore Corp, Bedford, Mass.), the emulsion was filled in vials
and stored at 4.degree. C. until used for intravenous
injection.
Example 9
5 mg/mL Paclitaxel Emulsion Formulation QWC
An additional emulsion of paclitaxel was prepared as described in
Example 8 but incorporating 5 instead of 10 mg/ml of the drug. The
composition of this emulsion is as follows:
TABLE-US-00006 paclitaxel 0.5 gm % .alpha.-tocopherol 6.0 gm % TPGS
3.0 gm % Poloxamer 407 1.0 gm % Sorbitol 4.0 gm % Triethanolamine
to pH 6.8 Water for injection qs to 100 mL
Following homogenization as described in Example 8, a somewhat
translucent emulsion of .alpha.-tocopherol and paclitaxel with a
mean particle diameter of 52 nm was obtained. Following sterile
filtration through a 0.22 micron Durapore filter (Millipore Corp.,
Bedford, Mass.), the emulsion was filled in vials and stored at
4.degree. C. until used for intravenous injection. Drug losses on
filtration were less than 1%.
Example 10
Paclitaxel Emulsion Formulation QWD
A fifth emulsion of .alpha.-tocopherol for intravenous
administration of paclitaxel was prepared as follows:
TABLE-US-00007 paclitaxel 0.5 gm % .alpha.-tocopherol 6.0 gm % TPGS
3.0 gm % Poloxamer 407 1.5 gm % Polyethyleneglycol 200 0.7 gm %
Sorbitol 4.0 gm % Triethanolamine to pH 6.8 Water for injection qs
to 100 mL
Synthetic .alpha.-tocopherol USP-FCC obtained from Roche Vitamins
(Nutley, N.J.) was used in this formation. Polyethyleneglycol 200
(PEG-200) was obtained from Sigma Chemical Co.
Following homogenization, a somewhat translucent emulsion with a
mean particle diameter of 60 nm was obtained. Following 0.22.mu.
sterile filtration through a 0.22 micron Durapore filter (Millipore
Corp, Bedford, Mass.), the emulsion was filled in vials and stored
at 4.degree. C. until used for intravenous injection. Drug losses
on filtration were less than 1%.
Example 11
Dissolution of Paclitaxel in TPGS and Preparation of Micellar
Solutions
We observed good solubility of paclitaxel in TPGS, about 100 mg
drug per 1.0 gm of TPGS. Micellar solutions of TPGS containing
paclitaxel were prepared as follows. A stock solution of paclitaxel
in TPGS was made up by dissolving 90 mg paclitaxel in 1.0 gm TPGS
at 45.degree. C. with ethanol, which was then removed under vacuum.
Serial dilutions were then prepared by diluting the paclitaxel
stock with additional TPGS to obtain paclitaxel in TPGS at
concentrations of 0.1, 1, 5, 10, 25, 50, 75 and 90 mg/mL. Using
fresh test tubes, 100 mg of each paclitaxel concentration in TPGS
was dissolved in 0.9 mL water. All test tubes were mixed by vortex
and by sonication at 45.degree. C. Clear micellar solutions in
water were obtained corresponding to final paclitaxel
concentrations of 0.01, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5 and 9.0
mg/mL.
A Nicomp Model 370 laser particle sizer (Particle Sizing Systems,
Santa Barbara, Calif.) was used to examine the solutions. Particle
sizes on the order of 10 nm were obtained, consistent with the
presence of micelles of TPGS and paclitaxel.
Micellar solutions of paclitaxel in TPGS containing up to 2.5 mg/mL
paclitaxel were stable for at least 24 hr, whereas those at 5.0,
7.5 and 9.0 mg/mL were unstable and drug crystals formed rapidly
and irreversibly. These observations imply that paclitaxel remains
solubilized only in the presence of an .alpha.-tocopherol-rich
domain within the emulsion particles. Thus, an optimum ratio of
.alpha.-tocopherol to TPGS is needed in order to produce emulsions
in which higher concentrations of paclitaxel can be stabilized.
When adjusted to the proper tonicity and pH, micellar solutions
have utility for slow IV drip administration of paclitaxel to
cancer patients, although the AUC is expected to be low.
The utility of TPGS in .alpha.-tocopherol emulsions is a synergy of
several desirable characteristics. First, it has its own affinity
for paclitaxel, probably by virtue of the .alpha.-tocopherol that
makes up the hydrophobic portion of its molecular structure.
Secondly, interfacial tension of TPGS in water with
.alpha.-tocopherol is about 10 dynes/cm, sufficient to emulsify
free .alpha.-tocopherol, especially when used with a co-surfactant.
Third, polyoxyethylated surfactants such as TPGS have
well-established, superior properties as a "stealth coat" for
injectable particles by dramatically reducing trapping of the
particles in the liver and spleen, as is well known in the art. But
the unexpected and unique finding with TPGS as a surfactant for
.alpha.-tocopherol emulsions was the finding of all three desirable
characteristics in a single molecule. An additional advantage of
TPGS is the fact that it forms stable self-emulsifying systems in
mixtures with oils and solvents such as propylene glycol and
polyethylene glycol, suggesting a synergy when used with
.alpha.-tocopherol for oral drug delivery.
When adjusted to the proper tonicity and pH, micellar solutions
have utility for slow IV drip administration of paclitaxel to
cancer patients, although the AUC is expected to be low.
Example 12
20 mg/mL Paclitaxel Emulsion Formulation
A coarse emulsion containing 20 mg/mL paclitaxel in
.alpha.-tocopherol was obtained with 5% .alpha.-tocopherol and 5%
TPGS by the methods described in Example 4, simply by increasing
the concentrations. No effort was made to test higher
concentrations simply because no further increase is necessary for
clinically useful intravenous emulsions.
Example 13
Use of Other PEG Surfactants in .alpha.-Tocopherol Emulsions
A variety of other pegylated surfactants, for example Triton X-100,
PEG 25 propylene glycol stearate, Brij 35 (Sigma Chemical Co), Myrj
45, 52 and 100, Tween 80 (Spectrum Quality Products), PEG 25
glyceryl trioleate (Goldschmidt Chemical Corp, Hopewell, Va.), have
utility in emulsifying .alpha.-tocopherol.
However, experiments with some other pegylated surfactants failed
to convincingly stabilize paclitaxel in an .alpha.-tocopherol
emulsion. To demonstrate the unique utility of TPGS, three
emulsions were prepared as described in Example 9, but Tween 80 and
Myrj 52 were substituted for TPGS as the primary surfactant in
separate emulsions. These two surfactants were chosen because Tween
80 and Myrj 52 have HLB values essentially equivalent to TPGS and
make reasonably good emulsions of .alpha.-tocopherol. However, when
5 mg/mL paclitaxel was included in the formulation, drug
crystallization was noted very rapidly after preparation of the
pre-emulsion, and the processed emulsions of Tween 80 and Myrj 52
were characterized as coarse, containing rod-shaped particles up to
several microns in length, consistent with crystals of paclitaxel.
Unlike the TPGS emulsion, which passed readily through a 0.22
micron filter with less than 1% loss of drug, the Tween and Myrj
emulsions were unfilterable because of the presence of this
crystalline drug material.
There are several possible explanations for the unexpected
improvement of the .alpha.-tocopherol paclitaxel emulsions with
TPGS. The drug has good solubility in TPGS, up to about 100 mg/mL.
Most likely it is the strength of the affinity of paclitaxel benzyl
side chains with the planar structure of the .alpha.-tocopherol
phenolic ring in the TPGS molecule that stabilizes the complex of
drug and carrier. In addition the succinate linker between the
.alpha.-tocopherol and PEG tail is a novel feature of this molecule
that distinguishes its structure from other PEGylated surfactants
tested.
Example 14
Poloxamer Based .alpha.-Tocopherol Emulsion
TABLE-US-00008 .alpha.-tocopherol 6.0 gm % Poloxamer 407 2.5 gm %
Ascorbyl Palmitate 0.3 gm % Sorbitol 6.0 gm % Triethanolamine to pH
7.4 Water qs to 100 mL
An .alpha.-tocopherol emulsion was prepared using Poloxamer 407
(BASF) as the primary surfactant. The white milky pre-mixture was
homogenized with continuous recycling for 10 minutes at 25 Kpsi in
a C5 homogenizer (Avestin, Ottawa Canada) with a feed temperature
of 45.degree. C. and a chiller loop for the product out set at
15.degree. C. A fine, sterile filterable emulsion of
.alpha.-tocopherol microparticles resulted. However, when this
formulation was made with paclitaxel, precipitation of the
paclitaxel was noted following overnight storage in the
refrigerator, again underlying the superior utility of TPGS as the
principle surfactant.
Example 15
Lyophilized Emulsion Formulation
Maltrin M100 (Grain Processing Corporation, Muscatine, Iowa) was
added as a 2.times. stock in water to the emulsion of Example 14.
Aliquots were then frozen in a shell freezer and lyophilized under
vacuum. On reconstitution with water, a fine emulsion was
recovered.
Lyophilized formulations have utility where the indefinite shelf
life of a lyophilized preparation is preferred. Lyophilizable
formulations containing other saccharides, such as mannitol,
albumin or PolyPep from Sigma Chemicals, St. Louis, Mo. can also be
prepared.
Example 16
In Vitro Release of Paclitaxel from .alpha.-Tocopherol
Emulsions
One of the desired characteristics of a drug delivery vehicle is to
provide sustained release of the incorporated drug, a
characteristic quite often correlated with improved
pharmacokinetics and efficacy. In particular, long-circulating
emulsions of paclitaxel can improve the delivery of the drug to
cancer sites in the body. We have surprisingly found that the
emulsions of the present invention do provide sustained release of
paclitaxel when compared to the only FDA-approved formulation of
paclitaxel at this time (Taxol.RTM., Bristol Myers Squibb (BMS),
Princeton, N.J. Emulsions were prepared having paclitaxel
concentrations of 6 mg/mL (QWA) and 7 mg/mL (QWB). For comparison,
Taxol contains 6 mg/ml of paclitaxel dissolved in
ethanol:cremophore EL 1:1 (v/v). In vitro release of paclitaxel
from the different formulations into a solution of
phosphate-buffered saline (PBS) at 37.degree. C. was monitored
using a dialysis membrane that is freely permeable to paclitaxel
(MW cut-off of 10 kilodaltons). Quantification of the drug in pre-
and post-dialysis samples was performed by HPLC. Drug release
profiles in terms of both percent release and concentration of
paclitaxel released over time were generated. As can be seen from
the data in FIG. 4, less than 5% of paclitaxel was dialyzed from
the emulsions over 24 hr, whereas about 12% was recovered outside
the dialysis bag from the marketed BMS formulation. This indicates
that drug release from the emulsion was significantly slowed
relative to the commercially available solution.
Example 17
Biocompatibility of .alpha.-Tocopherol Emulsions Containing
Paclitaxel
An acute single-dose toxicity study was performed. Mice 20 25 gm
each were purchased and acclimatized in an approved animal
facility. Groups of mice (n=3) received doses of the formulation
containing from 30 to 90 mg/kg paclitaxel in the .alpha.-tocopherol
emulsion prepared as described in Example 6. All injections were
given intravenously by tailvein bolus.
Although all injections were given by bolus IV push, no deaths or
immediate toxicity were observed at any dose, even at 90 mg/kg. The
results for body weight are shown in Table 2. Weight loss was 17%
in the highest group but all groups, even at 90 mg/kg, recovered or
gained body weight over a period of 10 days post injection.
A vehicle toxicity study was also done. Animals receiving drug-free
emulsion grew rapidly, and gained slightly more weight than animals
receiving saline or not injected. This was attributed to the
vitamin and calorie content of the formulation.
We observed a maximal tolerable dose (MTD) for paclitaxel of
greater than 90 mg/kg (Table 2), with no adverse reactions noted.
This is more than double the best literature values reported, in
which deaths were observed at much smaller doses. The FDA-approved
formulation of Taxol.RTM. causes death in mice at bolus intravenous
doses of 10 mg/kg, a finding repeated in our hands. In the rat,
Taxol.RTM. was uniformly fatal at all dilutions and dose regimes we
tested. In contrast, the composition of Example 6 was well
tolerated in rats, and is even improved over Taxotere, a less toxic
paclitaxel analogue commercially marketed by Rhone-Poulenc
Rorer.
One possible explanation for the high drug tolerance is that the
emulsion is behaving as a slow-release depot for the drug as
suggested from the in vitro release data in Example 16.
TABLE-US-00009 TABLE 2 Average Body Weight Change of Mice Treated
with Paclitaxel Emulsion Treatment Number of BW Change (gm) (dose,
mg/kg Animals Day 2 Day 7 Saline 4 1.0 3.4 Vehicle 4 1.2 3.5
Paclitaxel Emulsion 2 -1.0 2.2 (QWA) (36.3) Paclitaxel Emulsion 4
-1.8 1.7 (QWA) (54.4) Paclitaxel Emulsion 4 -1.5 1.6 (QWA) (72.6)
Paclitaxel Emulsion 1 -1.6 (QWA) (90.7)
Example 18
Efficacy Evaluation of Paclitaxel Emulsion
The paclitaxel emulsion of Example 6 was also evaluated for
efficacy against staged B16 melanoma tumors in nude mice and the
data is shown in Table 3. Once again, the marketed product
Taxol.RTM. was used as a reference formulation. Tumor cells were
administered subcutaneously and therapy started by a tail vein
injection at day 4 post-tumor administration at the indicated
dosing schedule. Efficacy was expressed as percent increase in
life-span (% ILS).
The following conclusions can be drawn from the data in Table 3: a)
an increased life span of about 10% was obtained by administration
of BMS Taxol at 10 mg/kg Q2Dx4, b) % ILS values improved to 30 50%
by administration of the .alpha.-tocopherol emulsion of paclitaxel
at 30, 40 or 50 mg/kg Q2Dx4, dose levels made possible by the
higher MTD, c) a nice dose response was observed when the emulsion
was administered at 30, 50 and 70 mg/kg Q4Dx3, with about 80% ILS
being observed at 70 mg/kg and, d) even at 90 mg/kg dosed only once
at day 4, there was about 36% ILS. These data clearly illustrate
the potential of the emulsions of the present invention to
substantially improve the efficacy of paclitaxel.
Example 19
Efficacy Evaluation of Paclitaxel Emulsions
The emulsions of Examples 6, 7 and 8 (QWA, QWB and QWC,
respectively) were compared for efficacy against B16 melanoma in
mice; Taxol.RTM. was again used as a reference formulation. Methods
essentially identical to those of Example 18 were used. The data
from this study is summarized in Table 3. Efficacy was expressed as
a) percent tumor growth inhibition (% T/C, where T and C stand for
treated and control animals, respectively); b) tumor growth delay
value (T-C), and c) log cell kill, which is defined as the ratio of
the T-C value over 3.32 .times.tumor doubling time. The latter
parameter for this particular tumor model was calculated to be 1.75
days. As can be seen from the results in Table 4, all measures of
efficacy, i.e., tumor growth inhibition, tumor growth delay value
and log cell kill, demonstrate superior efficacy of
.alpha.-tocopherol emulsions as a drug delivery vehicle over
Taxol.RTM., particularly when the emulsions were dosed every 4 days
at 70 mg/kg. As explained in Example 16, this increased efficacy is
likely a result of improved drug biocompatibility and/or sustained
release.
TABLE-US-00010 TABLE 3 Survival of Mice with B16 Tumors Treated
with QWA and Taxol .RTM. Mean Survival Time, Days % ILS.sup.b (vs
vehicle) Treatment Group & Schedule (Mean .+-. S.E.M.sup.a)
(Mean .+-. S.E.M) Vehicle Control (Days 4, 13.2 .+-. 0.9 -- 8, 12)
Saline Control (Days 4, 8, 15.8 .+-. 1.2 19.7 .+-. 8.6 12) Taxol
.RTM. (10 mg/kg) (Days 4, 16.4 .+-. 0.7 24.2 .+-. 5.4 6, 8, 10) QWA
(30 mg/kg) (Days 4, 6, 19.2 .+-. 1.4 45.4 .+-. 10.3 8) QWA (40
mg/kg) (Days 4, 6, 21.3 .+-. 1.4 61.4 .+-. 10.3 8) QWA (50 mg/kg)
(Days 4, 6, 18.8 .+-. 0.7 42.4 .+-. 5.7 8) QWA (30 mg/kg) (Days 4,
8, 15.3 .+-. 0.8 15.9 .+-. 6.4 12) QWA (50 mg/kg) (Days 4, 8, 20.7
.+-. 1.3 56.8 .+-. 9.5 12) QWA (70 mg/kg) (Days 4, 8, 24.2 .+-. 0.9
83.3 .+-. 6.4 12) QWA (90 mg/kg) (Day 4 only) 18.0 .+-. 0.6 36.4
.+-. 4.4 .sup.aSEM = Standard Error of Mean .sup.b% ILS = %
Increase in Lifespan = [(T - C) / C] .times. 100 where: T = mean
survival of treated C = mean survival of control
according to the NCI standards an ILS value greater than 50%
indicates significant anti-tumor activity.
TABLE-US-00011 TABLE 4 Comparison of 3 paclitaxel emulsions and
Taxol against early-stage B16 melanoma Median tumor Median % Log
Dosing Total wt. on day tumor wt. T/C cell Test Dosage Schedule
Dose 15 on day 18 Day T - C kill Article mg/kg/day (days) (mg/kg)
(mg) mg (range) 15 (days) total Control 0 4, 6, 8, 10 0 836 2139 --
-- -- Taxol .RTM. 20 4, 6, 8, 10 80 383 1217 46 2 0.34 QWA 20 4, 6,
8, 10 80 381 1197 46 2 0.34 QWA 40 4, 6, 8, 10 160 104 306 12 7 1.2
QWA 70 4, 8, 12, 16, 350 15 11 ~2 20 QWB 20 80 197 653 24 5 0.86
QWB 30 4, 6, 8, 10 120 139 449 17 5 0.86 QWB 40 4, 6, 8, 10 Toxic
QWC 20 80 319 848 34 3 0.52 QWC 40 4, 6, 8, 10 160 53 194 6 8 1.4
QWC 70 4, 6, 8, 10 350 33 66 4 >15 >2.6 4, 8, 12, 16, 20
Tumor Doubling Time calculated to be 1.75 days. % T/C = Tumor
Growth Inhibition (Day 15) = (median tumor wt. of treated/median
tumor wt. control) .times. 100 T - C = Tumor Growth Delay value =
median time for treatment group (T) and control group (C) tumors to
reach a predetermined size (usually 750 1000 mg) Log cell kill = (T
- C value)/(3.32 .times. tumor doubling time)
Example 20
Self-Emulsification of an .alpha.-Tocopherol/Tagat TO Mixture
.alpha.-tocopherol 2.0 gm and Tagat TO (Goldschmidt Chemical Corp,
Hopewell, Va.) 800 mg were dissolved together. About 80 mg of the
oily mixture was transferred to a test tube and water was then
added. With gentle hand mixing, there was immediate development of
a rich milky emulsion, consistent with "self-emulsifying systems"
proposed as drug delivery systems, in which surfactant-oil mixtures
spontaneously form an emulsion upon exposure to aqueous media.
Example 21
Self-Emulsifying Formulation Containing Paclitaxel
Paclitaxel 50 mg/ml was prepared in .alpha.-tocopherol by the
method described in Example 1. Tagat TO 20% (w/w) was added. The
resultant mixture was clear, viscous and amber in color. A 100 mg
quantity of the oily mixture was transferred to a test tube. On
addition of 1 mL of water, with vortex mixing, a fine emulsion
resulted.
Example 22
Self-Emulsifying Formulation of Paclitaxel
Paclitaxel 50 mg/ml was prepared in .alpha.-tocopherol by the
method described in Example 1. After removal of the ethanol under
vacuum, 20% TPGS and 10% polyoxyethyleneglycol 200 (Sigma Chemical
Co) were added by weight. A demonstration of the
self-emulsification ability of this system was then performed by
adding 20 mL of deionized water to 100 mg of the oily mixture at
37.degree. C. Upon gentle mixing, a white, thin emulsion formed,
consisting of fine emulsion particles demonstrated with the Malvern
Mastersizer (Malvern Instruments, Worcester, Mass.) to have a mean
size of 2 microns, and a cumulative distribution 90% of which was
less than 10 microns.
Example 23
Etoposide Emulsion Formulation in .alpha.-Tocopherol
Etoposide 4 mg (Sigma Chemical Co) was dissolved in the following
surfactant-oil mixture:
TABLE-US-00012 Etoposide 4 mg .alpha.-tocopherol 300 mg TPGS 50 mg
Poloxamer 407 50 mg
Ethanol and gentle warming was used to form a clear amber solution
of drug in oil. The ethanol was then removed under vacuum.
A pre-emulsion was formed by adding 4.5 mL of water containing 4%
sorbitol and 100 mg TPGS at 45.degree. C. with sonication. The
particle size was further reduced by processing in an Emulsiflex
1000 (Avestin, Ottawa Canada). The body of the Emulsiflex 1000 was
fitted with a pair of 5 mL syringes and the entire apparatus heated
to 45.degree. C. before use. The 5 mL of emulsion was then passed
through it by hand approximately 10 times. A free flowing,
practical emulsion of etoposide in an .alpha.-tocopherol vehicle
resulted.
We note that the solubilized form of etoposide in
.alpha.-tocopherol can also be used as an oral dosage form by
adaption of the methods of the preceding examples.
Example 24
Dissolution of Ibuprofen or Griseofulvin in .alpha.-Tocopherol
Ibuprofen is a pain-killer, and may be administered by injection
when required if there is danger that the drug will irritate the
stomach. The following solution of ibuprofen in .alpha.-tocopherol
may be emulsified for intravenous administration.
Ibuprofen (Sigma Chemicals), 12 mg crystalline, dissolved without
solvent in .alpha.-tocopherol, 120 mg, by gentle heating. The
resultant 10% solution of ibuprofen in vitamin E can be emulsified
by the methods described in Example 4, 6, 7, 8 or 22.
An antifungal compound, griseofulvin, 12 mg, was first dissolved in
3 mL of anhydrous ethanol; .alpha.-tocopherol was then added, 180
mg, and the ethanol was removed with gentle heating under vacuum.
The resultant solution of griseofulvin in .alpha.-tocopherol is
clear and can be emulsified by the methods described in Examples 4,
6, 7, 8 or 22.
Example 25
Vitamin E Succinate Emulsion Formulation
Vitamin E succinate has been suggested as a therapeutic for the
treatment of lymphomas and leukemias and for the chemoprevention of
cancer. The following is a composition and method for the
emulsification of vitamin E succinate in .alpha.-tocopherol.
Sucrose ester S1170 is a product of Mitsubishi Kagaku Foods Corp,
Tokyo, Japan. Vitamin E succinate, as the free acid, was obtained
as a whitish powder from ICN Biomedicals, Aurora, OH. Emulsions
incorporating other surfactants, such as pluronics, and TPGS along
with .alpha.-tocopherol and .alpha.-tocopherol succinate can be
prepared in a similar manner with and without a therapeutic
agent.
.alpha.-tocopherol 8 gm and vitamin E succinate 0.8 gm were
dissolved together in ethanol in a round bottom flask. After
removal of the solvent, 100 mL of an aqueous buffer was added, the
alkaline buffer consisting of 2% glycerol, 10 mM triethanolamine,
and 0.5 gm % sucrose ester S1170. After mixing for 2 mm, the
pre-emulsion was transferred to an Avestin Model C-5 homogenizer
and homogenization was continued for about 12 minutes at a process
feed temperature of 58.degree. C. The pressure differential across
the interaction head was 25 to 26 kpsi. During homogenization, the
pH was carefully monitored and adjusted as required to pH 7.0. Care
was taken to exclude oxygen during the process. A fine white
emulsion resulted.
Example 26
.alpha.-Tocopherol Levels in Esters
Levels of .alpha.-tocopherol in commercially available esters:
tocopherol-acetate, -succinate, -nicotinate, -phosphate and TPGS
were either provided by the vendor or determined by HPLC. The
concentration of free .alpha.-tocopherol in these solutions is less
than 1.0%, generally less than 0.5%.
Example 27
Resveratrol Emulsion Formulation
Resveratrol is a cancer chemopreventative first discovered as an
extract of grape skins. It has been proposed as a dietary
supplement.
Resveratrol was obtained from Sigma Chemical Co. While it dissolved
poorly in ethanol, upon addition of 10 mg resveratrol, 100 mg of
.alpha.-tocopherol, 100 mg TPGS and ethanol, a clear solution
formed rapidly. Upon removal of the ethanol, a clear amber oil
remained.
The oily solution of resveratrol can be formulated as a
self-emulsifying system for oral delivery by the various methods of
the preceding examples.
Example 28
Muramyl Dipeptide Formulation
Muramyl dipeptides are derived from mycobacteria and are potent
immunostimulants representative of the class of muramyl peptides,
mycolic acid and lipopolysaccharides. They have use, for example,
in the treatment of cancer, by stimulating the immune system to
target and remove the cancer, particularly in connection with
anti-cancer vaccines. More recently, muroctasin, a synthetic
analog, has been proposed to reduce non-specific side effects of
the bacterial wall extracts.
N-acetylmuramyl-6-O-steroyl-1-alanyl-d-isoglutamine was purchased
from Sigma Chemical Co. and 10 mg was dissolved in 100 mg
.alpha.-tocopherol and 80 mg TPGS. Ethanol was used as a co-solvent
to aid in dissolution of the dipeptide, but was removed by
evaporation under vacuum, leaving a clear solution in
.alpha.-tocopherol and surfactant.
This oil solution of the drug can be emulsified for parenteral
administration by the various methods of the preceding
examples.
Example 29
Alcohol-Containing Emulsion
In attempting to adapt the teachings of PCT WO 95/11039 to the oral
administration of paclitaxel, the following formulation was
made.
TABLE-US-00013 paclitaxel 0.125 gm .alpha.-tocopherol 0.325 gm TPGS
0.425 gm Ethanol 0.125 gm
As before, paclitaxel was dissolved in a .alpha.-tocopherol and
TPGS with ethanol, which was then removed under vacuum. By dry
weight, residual ethanol was less than 3 mg (0.3% w/w). Fresh
anhydrous ethanol 0.125 gm was then added back to the formulation.
After mixing, the suitability of the formulation for oral
administration, as in a gelatin capsule, was simulated by the
following experiment. An aliquot of 100 mg of the free-flowing oil
was added to 20 mL of water at 37.degree. C. and mixed gently with
a vortex mixer. A fine emulsion resulted. But after twenty minutes,
microscopy revealed the growth of large numbers of crystals in
rosettes, characteristic of paclitaxel precipitation. It was
concluded that this formulation was not suitable for oral
administration of paclitaxel because large amounts of the drug
would be in the form of crystals on entry into the duodenum, where
it would be prevented from uptake because of its physical form. We
speculate that the excess of ethanol, in combination with the high
ratio of TPGS to .alpha.-tocopherol, is responsible for the
observed crystallization of the drug from this formulation.
Example 30
Alcohol-Containing .alpha.-Tocopherol Emulsion
In attempting to adapt the teachings of PCT WO 95/11039 to the
intravenous administration of paclitaxel, the following formulation
was made:
TABLE-US-00014 paclitaxel 0.050 gm .alpha.-tocopherol 0.100 gm
Lecithin 0.200 gm Ethanol 0.100 gm Butanol 0.500 gm
As before, paclitaxel was dissolved in .alpha.-tocopherol and TPGS
with ethanol, which was then removed under vacuum. By dry weight,
residual ethanol was less than 2 mg (0.5% w/w). Fresh anhydrous
ethanol 0.100 gm and n-butanol 0.500 gm were then added back to the
formulation. A clear oil resulted. The injection concentrate was
tested for biocompatibility in administration by the standard
pharmaceutical practice of admixture with saline. About 200 mg of
the oil was placed into 20 mL of saline and mixed. Large flakes of
insoluble material developed immediately and the greatest amount of
material formed dense deposits on the walls of the test tube. The
mixture was clearly unsuitable for parenteral administration by any
route, and we speculate that this is so regardless of the identity
of the drug contained in the formulation. By trial and error we
have learned that lecithin is a poor choice as a surfactant for
.alpha.-tocopherol by virtue of its low HLB (around 4). Other
successful examples described here for fine emulsions suitable for
parenteral administration were all made with high HLB surfactants.
These surfactants include TPGS (HLB around 17), Poloxamer 407 (HLB
about 22) and Tagat TO (HLB about 14.0). In general, we found that
.alpha.-tocopherol emulsification is best performed with principal
surfactants of HLB>10, preferably greater than 12. Lecithin is
not in this class, although it could be used as a co-surfactant. In
comparison, typical 0/w emulsions of triglycerides are made with
surfactants of HLB between 7 and 12, demonstrating that
.alpha.-tocopherol emulsions are a unique class by virtue of the
polarity and extreme hydrophobicity of the .alpha.-tocopherol,
factors that also favor the solubility of lipophilic and slightly
polar lipophilic drugs in .alpha.-tocopherol. See Emulsions: Theory
and Practice, 2d ed., p. 248 (1985).
Example 31
Various formulations useful in the invention (Table 5) are prepared
as follows:
TABLE-US-00015 TABLE 5 B A (all surfactant in (split surfactant)
oil) Composition of Injectable Weight Weight Weight Weight
Paclitaxel Emulsions (g) (%) (g) (%) Oil Phase Paclitaxel 0.50 0.51
0.53 0.52 PEG 400 6.02 6.04 6.38 6.30 TPGS 3.78 3.80 5.32 5.25
Pluronic 1.07 1.05 F127 Vitamin E 8.04 8.07 8.51 8.40 Aqueous Phase
TPGS 1.25 1.26 Pluronic 1.01 1.01 F127 Water 79.00 79.31 79.50
78.48 Total 99.60 100.00 101.30 100.00
Formulation A--Split Surfactants: 1) 1.25 g TPGS and 1.01 g
Pluronic F127 were dissolved in 79.00 g water for injection by
heating and stirring. 2) 0.533 g paclitaxel was dissolved in 6.354
g PEG 400 by mixing (low shear) at 75.degree. C. 3) 3.992 g TPGS
and 8.490 g Vitamin E were added and mixed (low shear) at
45.degree. C. until TPGS was melted and the mixture was visibly
homogeneous. This oil phase represents a slight excess in order to
account for incomplete transfer in Step 4. 4) The aqueous phase
(step 1) was heated to 45.degree. C. and mixed at medium shear
(laboratory mixing motor) while 45.degree. C. oil phase (step 2+3)
was poured in over 1 minute. Mixing was continued 2 minutes more to
form a crude emulsion. 5) The emulsion was homogenized in an
Avestin C5 in continuous recycle mode for 1 hour at 22 Kpsi peak
stroke pressure. 6) Actual amounts and percentages shown in the
table are corrected for the incomplete transfer of oil phase during
Step 4.
This method utilizing the split surfactants is useful in the cases
where the solubility of a particular surfactant in the oil phase is
limited.
Formulation B--All Surfactants in Oil Phase
1) 1.066 g paclitaxel was dissolved in 12.887 g PEG400 by mixing
(low shear at 75.degree. C. 2) 10.739 g TPGS and 2.157 g Pluronic
F127 were added and mixed (low shear) at 50 60.degree. C. until
both surfactants were completely melted/dissolved. 3) 17.176 g
Vitamin E was added and mixed (low shear) at 45 50.degree. C. until
the mixture was visibly homogeneous. 4) 21.8 g of the oil phase
produced in Steps 1 4 was added over 1 minute to 79.5 g water while
mixing at medium shear (laboratory mixing motor). Mixing was
continued for a total of 3 minutes to form a crude emulsion. 5)
Emulsion was homogenized in an Avestin C5 in continuous recycle
mode for 30 minutes at 22 K psi peak stroke pressures.
From a processing perspective it is advantageous to have all of the
surfactants in the oil phase. Both the dispersion of the
pre-emulsion and subsequent homogenization are facilitated and
potential gellation of high melting point surfactants, such as
TPGS, can be avoided.
Example 32
Etoposide Emulsion
A vitamin E emulsion (6.0% vitamin E, 3.5% TPGS, 6.0%, PEG400, 8%
Pluronic F-127) and incorporating 2 mg/ml of Etoposide was prepared
as follows: 1) 0.1044 g of Etoposide was dissolved in 3.1435 g of
PEG 400 (5 min at 65.degree. C.). 2) 2.0447 g of TPGS and 3.1563 g
of Vitamin E were added and mixed until complete dissolution. 3)
The oil phase was mixed at 44.degree. C. with 42.4 g of water for
injection incorporating 0.5 g of Pluronic F-127 (the aqueous phase
was degassed by boiling prior to its mixing with the oil phase) and
the pre-emulsion was formed by brief sonication. 4) Upon
homogenization in an Avestin C5 at 22 24 Kpsi a fine emulsion was
formed.
Example 33
Etoposide Emulsion
An .alpha.-tocopherol emulsion containing PEG 300 and incorporating
2mg/ml of Etoposide was prepared as follows:
Etoposide was first dissolved in PEG-300 (10 min at 72.degree. C.).
TPGS and Vitamin E were then added to the drug solution. Aqueous
phase (WFI containing Poloxamer 407) was degassed by boiling prior
to use. Pre-emulsion was prepared by adding 5 g of the oil phase to
45 g of water at 45.degree. C. After a 3-min mixing the
pre-emulsion was homogenized at 25 Kpsi for 30 min to produce a
fine emulsion. The final composition of the emulsion is shown
below:
TABLE-US-00016 Component Composition (%, w/w) Etoposide 0.2 Vitamin
E 3.0 TPGS 1.5 PEG-300 3.0 Poloxamer 407 1.0 WFI (water for
injection) 92.3
Example 34
Additional paclitaxel emulsions for injection are presented in
Table 6.
TABLE-US-00017 TABLE 6 Composition of Injectable Paclitaxel
Emulsions D E C (all surfactant in (all surfactant in Composition
of (split surfactant) oil) oil) Injectable Paclitaxel Weight Weight
Weight Weight Weight Weight Emulsions (g) (%) (g) (%) (g) (%) Oil
Phase Paclitaxel 2.0 0.4 0.55 1.1 0.5 0.5 PEG 400 32.0 6.4 3.36 6.7
10.0 10.0 TPGS 18.85 3.77 2.60 5.2 4.3 4.3 Pluronic 0.52 1.0 5.1
1.1 F127 Vitamin E 40.5 8.1 4.19 8.4 7.2 7.2 Aqueous TPGS 6.4 1.28
Phase Pluronic 5.0 1.0 F127 Water 395.25 79.05 41.0 82.0 79.5 79.5
Total 500.0 100.0 52.2 104.4 102.6 102.6
Table 6. Composition of Injectable Paclitaxel Emulsions
Example 35
Compositions of various self-emulsifying emulsions useful in this
invention are shown in Table 7.
TABLE-US-00018 TABLE 7 Self-Emulsifying Emulsions Composition of
SEFP-1 SEFP-2 Self-Emulsifying Weight Weight Weight Weight
Emulsions (g) % (g) % Paclitaxel 0.255 5.11 0.258 5.14 Vitamin E
1.989 19.88 2.242 44.70 TPGS 0.992 19.99 0.765 15.25 PEG 400 1.502
30.11 0.999 19.92 Pluronic F127 0.250 5.01 Solutol HS15 0.752 14.99
Total 4.988 100.00 5.016 100.00
The emulsions described in Table 7 were synthesized as follows.
SEFP-1
Paclitaxel and PEG 400 were heated together at 60 67.degree. C. and
stirred until the drug was dissolved in PEG (15 min). Then TPGS and
Pluronic F127 were added and stirred at 70.degree. C. for 10 15 min
to dissolve the surfactant. Finally, Vitamin E (.alpha.-tocopherol)
was added and mixed for 5 10 min at 55.degree. C. until the mixture
was clear and homogeneous. Upon dilution with an aqueous phase a
fine emulsion can be obtained.
SEFP-2
Paclitaxel and PEG 400 were first stirred at 65 75.degree. C. for
45 min, then TPGS was added and stirring was continued for another
30 mm to completely dissolve all three components and produce a
clear solution. Finally, Solutol HS-15 and vitamin E were added and
mixed for about 5 mm at 55.degree. C. to obtain a clear homogeneous
liquid. Upon dilution with an aqueous phase, a fine emulsion can be
obtained.
Example 36
Additional compositions of self-emulsifying emulsions of paclitaxel
are shown in Table 8.
TABLE-US-00019 TABLE 8 Self-Emulsifying Emulsions Composition of
Self- SEFP-3 SEFP-4 Emulsifying Weight Weight Weight Weight
Emusions (g) (%) (g) (%) Paclitaxel 0.10 2 0.05 1
.alpha.-Tocopherol 1.40 28 0.50 10 TPGS 1.00 20 0.95 19 PEG400 1.00
20 1.00 20 Solutol HS-15 1.50 30 2.50 50 Total 5.00 100 5.00
100
SEPF-3 and SEFP-4 were prepared by first dissolving paclitaxel in
solutol HS-15+PEG 400 by low shear mixing at 60 70.degree. C.
(<30 mm). TPGS and .alpha.-tocopherol were then added and
briefly mixed to form a clear solution (TPGS solidifcation can be
observed at room temperature but remains a clear liquid at
37.degree. C.).
The particle size of the emulsions upon dilution of SEFP-3 and
SEFP-4 was determined as follows: 0.2 ml of SEFP-3 or SEFP-4 was
diluted in 100 ml of phosphate-buffered saline at 37.degree. C. by
low shear mixing with a stir bar for 5 minutes. An emulsion was
quickly formed, the particle size of which was measured by the
Malvern Mastersizer. The volume mean diameter of SEFP-3 and SEFP-4
was found to be 2.49 and 1.55 .mu.m, respectively.
For an efficient self-emulsified system the mean droplet diameter
of the resulting emulsion should be less than 10 .mu.m and
preferably less than 5 .mu.m.
Example 37
Paclitaxel Emulsions Incorporating a PEGylated Phospholipid
DMPE-PEG.sub.2000 (Dimyristoyl Phosphatidyl Ethanolamine
Polyethylene Glycol 2000) incorporating emulsions were prepared
(Table 9). Paclitaxel, when present, was first dissolved in PEG 400
by low shear mixing at 75.degree. C. The other ingredients were
added and briefly mixed (after melting TPGS, and in the ease of
DMPEG-2, the P 407) to form a clear solution. A vacuum was applied
to degas the oil phase prior to emulsification, and the oil phase
was brought to 45.degree. C. Water was boiled for 15 minutes to
degas, then brought to 45.degree. C. also. The two phases were
mixed at 45.degree. C. at low to medium shear to form a
pre-emulsion. For formulations DMPE-PEG-P2, DMPE-PEG-P3 and
DMPE-PEGP-4, this was accomplished by simply adding the warm water
to the oil phase and swirling by hand with sonication. The
pre-emulsion for DMPE-PEG2 was prepared by pouring the oil phase
into water while stirring with a laboratory mixing motor.
Pre-emulsions were immediately homogenized in the Avestin C5
homogenizer at 20 22K psi peak stroke pressure to produce fine
emulsions with a mean droplet diameter and 99% cumulative
distributions of less than 200 nm.
TABLE-US-00020 TABLE 9 Paclitaxel Emulsions Incorporating a
Pegylated Phospholipid DMPE- DMPE- DMPE- DMPE- PEG-1 PEG-2 PEG-3
PEG-4 (g) (%) (g) (%) (g) (%) (g) (%) Paclitaxel 0.53 1.1 0.96 1.0
PEG 400 3.07 6.1 5.77 5.8 1.8 6.0 1.84 6.1 TPGS 2.59 5.1 4.62 4.7
1.51 5.0 1.22 4.1 DMPE-PEG.sub.2000 0.53 1.1 0.20 0.2 0.30 1.0 0.62
2.1 Poloxamer 407 0.96 1.0 Vitamin E 4.11 8.2 7.71 7.8 2.42 8.1
2.14 7.2 Water 39.50 78.5 79.00 79.6 24.0 79.9 24.1 80.5 Total
50.33 100.0 99.23 100.0 30.03 100.0 29.92 100
Example 38
Efficacy Data
Formulation D (Table 6) was evaluated for efficacy against B16
melanoma in mice as described in Examples 18 and 19 and the data is
summarized in FIG. 5. Comparative efficacy data is presented in
Table 10.
TABLE-US-00021 TABLE 10 Comparative Efficacy in B16 Melanoma Tumor
Model: Taxol .RTM. vs SONUS Paclitaxel Emulsion "QW 8184" Total % %
Dosage Dose Mortal- T/C Log Test (mg/kg/ Schedule (mg/ Median Tumor
Weight on Day ity (by Day T - C cell Article day) (days) kg) 1 4 7
10 13 17 day 17) 13 (days) kill Saline 80 q4dx5 -- 8 245 1271 1800
2916 14114 60 -- -- -- equiv. 0 Taxol .RTM. 20 qdx5 100 6 123 331 2
2192 4901 20 75 5 0.9 9 Formu- 60 q3dx5 300 1 106 221 234 400 400
60 14 13 2.3 lation 0 D 8 % T/C = Tumor Growth Inhibition (median
tumor wt. of treated/median tumor wt. control) .times. 100 T - C =
Tumor Growth Delay value (median time for treatment group (T) and
control group tumors (C) to reach a predetermined size (>750 mg)
Log cell kill = (T - C value)/(3.32 .times. tumor doubling time)
tumor doubling time calculated to be 1.75 days.
Consistent with the data in Table 4, efficacy assessment by tumor
growth inhibition, tumor growth delay and log cell kill indicate
significant improvement with Formulation D over Taxol.RTM..
Example 39
Physical Stability Data
The physical stability of formulation D was assessed by potential
particle size changes upon storage and the data is shown in Table
10.
TABLE-US-00022 TABLE 11 Physical Stability of Formulation D
Volume-weighted Particle Size (nm) Storage Day Mean Droplet
Distribution 99% of (2 8.degree. C.) Diameter the Particles less
than 2 71.3 154.6 3 69.3 151.8 10 67.7 151.6 15 69.8 150.8 28 66.3
152.3 30 66.9 150.3
Particle size was measured using the Nicomp 370 sub-micron particle
size analyzer. As can be seen from the data in Table 11 no
significant changes were observed in either the mean droplet
diameter or the 99% cumulative distribution of the particles. The
latter parameter is often used as an indicator of particle
aggregation and growth. In addition, no precipitation or other
gross changes were observed during storage. Long term stability is
ongoing.
Example 40
Chemical Stability
The chemical stability of formation D (Table 6) was monitored by
HPLC using the procedures of Example 5 and the data is shown in
Table 12. HPLC is utilized to quantitate the concentration of
paclitaxel and degradants. In Table 12, drug concentration is
equivalent to drug potency.
TABLE-US-00023 TABLE 12 Chemical Stability of Formulation D Storage
Day Drug Concentration (2 8.degree. C.) (mg/ml) 0 9.53 10 9.54 19
9.39 32 9.54
It is evident from this data that the drug potency in formulation D
remains unchanged under these storage conditions.
In addition, no degradation of the drug was observed during this
storage time.
Example 41
Emulsions Containing PEG 300 or NMP
.alpha.-tocopherol emulsions containing PEG 300 or NMP
(N-Methyl-2-pyrrolidone) and incorporating 10 mg/ml paclitaxel are
shown in Table 13.
TABLE-US-00024 TABLE 13 PEG 300 NMP Weight Weight Weight Weight
Component (g) % (g) % Paclitaxel 0.05 1.0 0.05 1.0 PEG 300 0.32 6.2
NMP (N-Methyl-2- 0.18 3.6 pyrrolidone) TPGS 0.25 4.9 0.25 5.0
Poloxamer 0.05 0.9 0.05 1.0 407 Vitamin E 0.40 7.9 0.43 8.7 Water
4.00 78.9 4.00 80.7 Total 5.07 100.0 4.96 100.0
In both cases, paclitaxel was first dissolved in the solvent (PEG
300 or NMP) with low shear mixing. Heating to 60.degree. C. was
used with the PEG 300 to speed the dissolution while with the NMP
formulation a few minutes at room temperature was sufficient to
dissolve the drug. The remaining ingredients (except water) were
then added and the mixtures were heated to 60 65.degree. C. with
low shear mixing to melt the solid surfactant and produce
homogeneous, clear solutions. The solutions were brought to
45.degree. C., then 45.degree. C. water was added to them. The
resulting mixtures were processed under medium shear to produce a
thick, white crude emulsion, very similar in appearance to the
pre-emulsion of formulation D (Table 6). These emulsions can
further be homogenized at high pressure to produce fine
emulsions.
Example 42
Large Scale Preparation of Formulation D (QW 8184)
Using procedures analogous to those described in previous examples,
formulation D (Table 6) was manufactured at a large scale in
2.times.2L sub-lots having the following composition:
TABLE-US-00025 TABLE 14 Sub-Lot 1 Sub-Lot 2 Amount in Amount in Oil
Phase Weight Oil Phase Weight Component (g) (%) (g) (%) Paclitaxel
21 1.01 21 1.01 PEG400 123.6 5.96 123.6 5.92 .alpha.-Tocopherol
164.8 7.94 164.8 7.89 TPGS 103 4.97 103 4.93 Poloxamer 407 20.6
0.99 20.6 0.99 Oil Phase 433 20.97 413.2 20.73 Total
For the preparation of the pre-emulsion, 416.8 g of the oil phase
of sub-lot 1 and 413.2 g of the oil phase of sub-lot 2 were mixed
with 1580 g of water for injection (5 min at 46.degree. C.). Upon
homogenization fine emulsions were produced having a mean droplet
diameter of about 70 nm, that is, very similar to that of
formulation D at the small scale (Table 10). This scaled
formulation was further sterilized by filtration through a 0.2
micron filter.
Example 43
Hemolytic Activity Evaluation of a Drug-Free Emulsion
A large scale (2.5 L) of formulation D in the absence of paclitaxel
was prepared as described in Example 42 having the following
composition.
TABLE-US-00026 TABLE 15 Amount in Oil Phase Component (g) Weight %
PEG400 154.5 5/97 .alpha.-Tocopherol 206 7.96 TPGS 128.8 4.97
Poloxamer 407 25.8 1.00 Oil Phase 515.1 19.89 Total
For the preparation of the pre-emulsion, 496.7 g of the oil phase
were mixed with 2000 g of water for injection (5 min at 46.degree.
C.). Upon homogenization and filter sterilization this formulation
was evaluated for gross hemolytic reaction with human blood using
the following procedure:
Volunteer healthy blood was collected with heparin by Vacutainer
stick. The plasma was initially straw colored and negative for
hemolysis. Drops of whole blood and the drug-free emulsion were
brought together under coverslip and observed microscopically for
several minutes. During contact, red blood cells (RBCs) remained
normocytic. No obvious aggregation of the emulsion particles was
noted. No gross changes in platelet or WBC morphology were noted.
Then, in test tubes, whole blood and vehicle were mixed 1:1 and
5:1, v/v. As a control, whole blood was mixed with saline for
injection 1:1. All mixtures were incubated at 37.degree. C. and
examined at 10 and 30 min. Supernatants in all three tubes were
straw colored and clear. It can be concluded form this study that
there is no immediate gross hemolytic reaction between the emulsion
vehicle and blood. This suggests that the morphology of the red
cell membranes is not perturbed by the surfactants present in the
emulsion, in contrast to several reports in the literature on
surfactant-induced hemolysis of RBC.
Example 44
Physical Stability Data
Table 16 shows long-term stability of the scaled up formulation of
Example 42 upon a 9month storage at 4.degree. C. or 25.degree. C.
It is evident that at least during this storage time, both the mean
droplet diameter and the 99% cumulative distribution did not
significantly change from their initial values of about 65 and 150
nm, respectively, and the emulsion remains within
specifications.
TABLE-US-00027 TABLE 16 Physical Stability of QW8184 99% Cumulative
Storage Mean Droplet Diameter, Distribution, nm Time nm (mean .+-.
sd) (mean .+-. sd) (months) 4.degree. C. 25.degree. C. 4.degree. C.
25.degree. C. 0.0 64 .+-. 0.8 63 .+-. 2.1 150 .+-. 0.7 150 .+-. 0.7
0.5 67 .+-. 2.9 63 .+-. 2.5 152 .+-. 2.8 149 .+-. 3.6 1.1 64 .+-.
2.5 65 .+-. 2.5 149 .+-. 2.0 152 .+-. 2.1 3.1 66 .+-. 1.2 62 .+-.
2.0 150 .+-. 1.2 148 .+-. 2.5 6.1 63 .+-. 1.2 64 .+-. 3.1 150 .+-.
1.5 152 .+-. 4.0 9.2 64 .+-. 2.1 62 .+-. 1.0 152 .+-. 2.1 153 .+-.
0.7
Example 45
Chemical Stability
A 9-month chemical stability data of the scaled up formulation of
Example 42 in terms of paclitaxel potency and levels of known
degradants as shown in Tables 17 and 18. As can be seen from these
results, there were no significant changes in either the drug
potency or the levels of known degradants and the product remains
within specifications at both storage temperatures.
TABLE-US-00028 TABLE 17 Paclitaxel Potency and Degradants at
4.degree. C. Degradants Paclitaxel (%, mean .+-. sd, Storage
Potency n = 3) Time mean .+-. sd, n = 3 7-Epi- 10-Deacetyl-
(months) (mg/ML) paclitaxel Baccatin-3 paclitaxel 0.0 8.22 .+-.
0.64 0.17 .+-. 0.01 0.12 .+-. 0.01 0.15 .+-. 0.01 0.5 9.48 .+-.
0.08 0.32 .+-. 0.05 0.15 .+-. 0.00 0.16 .+-. 0.00 1.1 8.79 .+-.
0.53 0.31 .+-. 0.03 0.17 .+-. 0.00 0.17 .+-. 0.00 3.1 9.50 .+-.
0.07 0.61 .+-. 0.03 0.20 .+-. 0.00 0.20 .+-. 0.00 6.1 9.27 .+-.
0.17 0.28 .+-. 0.02 0.17 .+-. 0.01 0.18 .+-. 0.02 9.2 9.21 .+-.
0.12 0.36 .+-. 0.02 0.17 .+-. 0.00 0.18 .+-. 0.01
TABLE-US-00029 TABLE 18 Paclitaxel Potency and Degradants at
25.degree. C. Degradants Paclitaxel (%, mean .+-. sd, Storage
Potency n = 3) Time mean .+-. sd, n = 3 7-Epi- 10-Deacetyl-
(months) (mg/ML) paclitaxel Baccatin-3 paclitaxel 0.0 8.22 .+-.
0.64 0.17 .+-. 0.01 0.12 .+-. 0.01 0.15 .+-. 0.01 0.5 9.10 .+-.
0.65 0.33 .+-. 0.00 0.17 .+-. 0.00 0.17 .+-. 0.01 1.1 8.06 .+-.
0.75 0.32 .+-. 0.04 0.17 .+-. 0.00 0.17 .+-. 0.01 3.1 9.19 .+-.
0.79 0.65 .+-. 0.05 0.22 .+-. 0.00 0.22 .+-. 0.00 6.1 9.11 .+-.
0.71 0.33 .+-. 0.02 0.16 .+-. 0.02 0.15 .+-. 0.03 9.2 9.02 .+-.
0.68 0.36 .+-. 0.02 0.18 .+-. 0.01 0.18 .+-. 0.01
Example 46
Efficacy Evaluation
The formulation of Example 42 was evaluated for efficacy against
B16 melanoma as described in Examples 18, 19 and 38 and the results
are summarized in Table 19.
TABLE-US-00030 TABLE 19 Antitumor Activity of QW8184 vs Taxol .RTM.
in the B16 Melanoma Model Dose Survival Test mg/kg Schedule (mean
.+-. SD) % T/C.sup.a % TGI.sup.b T - C.sup.c Log Cell Article n = 8
Days days day 20 day 20 days Kill.sup.d Saline Control q3dx5 17
.+-. 2 -- -- -- -- Vehicle Control q3dx5 20 .+-. 1 93 3 3 -- Taxol
.RTM. 20 q3dx5 19 .+-. 5 77 23 3 0.5 QW8184 20 q3dx5 28 .+-. 7 11
89 10 1.8 QW8184 40 q3dx5 33 .+-. 5 0 100 17 3.0 .sup.a% T/C =
(Median Tumor Wt of treated/Median Tumor Wt of control) .times. 100
.sup.b% TGI = 100 - (% T/C) .sup.cT - C = Tumor Growth Delay Value
(median time for the treatment group (T) and control (C) to reach a
predetermined size (>750 mg) .sup.dLog Cell Kill = (T - C
value)/(3.32 .times. tumor doubling time)
By all end points of efficacy, QW8184 exhibited superior antitumor
activity in mice at doses that included or well exceeded the MTD of
Taxol.RTM. but which were well tolerated. Such effects have not
been reported with previous injectable emulsions of paclitaxel. MTD
is the maximum tolerated dose that is determined from acute
toxicity studies.
Example 47
Efficacy Evaluation
The antitumor activity of QW8184 (Example 42) was evaluated for
efficacy against the human ovarian tumor xenograft IGROV-1 using
the marketed product Taxol.RTM. as a reference formulation. Nude
mice were implanted subcutaneously by trocar with fragments of
IGROV-1 human ovarian carcinomas harvested from subcutaneously
growing tumors in nude mice hosts. When tumors were approximately
5.times.5 mm in size, the animals were pair matched into treatment
and control groups containing 9 ear-tagged tumor-bearing mice per
group. QW8184 was administered i.v. on q3dx5, q4dx5, and q5dx5
schedule at 20, 40, and 60 mg/kg. Taxol.RTM. was administered i.v.
on the same schedules at 20 mg/kg, its maximum tolerated dose. Mice
were weighed twice weekly, and tumor measurements were taken by
calipers starting Day 1 and converted to mg tumor weight. The
experiment was terminated when the control tumors reached
approximately 1 gr and tumors were excised and weighed and the mean
tumor weight per group was calculated. The data is summarized in
Table 20.
TABLE-US-00031 TABLE 20 Antitumor Activity of QW8184 vs Taxol .RTM.
in the IGROV-1 Human Ovarian Tumor Xenograft Mice with Dose Final
Tumor Wt Complete Group Schedule (mg/kg) (Mean .+-. SEM, mg) % TGI
Shrinkage Saline q3dx5 control 874.8 .+-. 178.6 -- 0 QW8184 q3dx5
vehicle 839.9 .+-. 80.4 4.4 0 QW8184 q3dx5 20 115.9 .+-. 39.1 93.4
2 QW8184 q3dx5 40 0.1 .+-. 0.1 -- 8 QW8184 q3dx5 60 0.0 .+-. 0.0 --
7 QW8184 q4dx5 20 69.2 .+-. 28.4 99.9 3 QW8184 q4dx5 40 0.0 .+-.
0.0 -- 9 QW8184 q4dx5 60 4.9 .+-. 4.9 -- 8 QW8184 qdx5 20 158.2
.+-. 56.7 88.7 3 Taxol .RTM. q3dx5 20 22.3 .+-. 14.2 -- 3 Taxol
.RTM. q4dx5 20 24.0 .+-. 11.5 -- 3 Taxol .RTM. qdx5 20 16.7 .+-.
9.6 -- 2
Administration of QW8184 at 20, 40 and 60 mg/kg on a q3dx5 or q4dx5
schedule resulted in nearly 100% tumor growth inhibition at all
doses with 2, 8, and 7 and 3, 9, and 8 complete tumor responses,
respectively. In comparison, administration of Taxol.RTM. resulted
in 3 complete tumor responses on both schedules. On a qdx5
schedule, the antitumor activities of QW8184 and Taxol.RTM. were
similar. QW8184, however, was better tolerated with no toxic deaths
whereas six toxic deaths were noted with Taxol.RTM.. QW8184 was
highly active against the IGROV-1 human ovarian xenograft model in
a dose-dependent fashion, regardless of the dosing schedule and it
was better tolerated than Taxol.RTM..
Example 48
Pharmacokinetic Study
The pharmacokinetics of the formulation of Example 42 (QW8184) in
the rat upon a single 10 mg/kg i.v. administration was determined
using Taxol.RTM. as a reference formulation. The drug was
administered iv. to male or female rats either as a 3-hr infusion
(Taxol.RTM.) or as a bolus dose (QW8184). Blood samples were
collected from 0 72 hrs after dose administration, plasma was
prepared by centrifugation and analyzed for paclitaxel
concentration using a high performance liquid chromatography (HPLC)
method with LC/MS/MS detection. Pharmacokinetic analysis was
performed on the mean composite plasma concentration-time profiles
using a model independent method. The derived pharmacokinetic
parameters are shown in Table 21. The pharmacokinetic parameters
determined were as follows:
T.sub.max: time required to reach peak plasma levels
(C.sub.max)
C.sub.max: peak plasma concentration of the drug
AUC.sub.O-t: non-extrapolated area under the plasma
concentration-time curve from time zero to time t which is the end
of the plasma sample collection
AUC.sub.0-.infin.: extrapolated area under the plasma
concentration-time curve from time zero to infinite
TABLE-US-00032 TABLE 21 Derived Pharmacokinetic Parameters of
Paclitaxel Following Intravenous Administration of QW8184 or Taxol
.RTM. in Rats at 10 mg/kg (70 mg/m.sup.2) Pharmaco- kinetic QW8184
Taxol .RTM. Parameter Male Female Male Female T.sub.max (hr) 0.083
0.083 3 3 C.sub.max (ng/mL) 58950 53900 5867 7227 AUC.sub.0 t 35504
32761 18138 22701 (ng hr/mL) AUC.sub.0 .infin. 35551 32829 18347
23002 (ng hr/mL) K.sub.e (hr.sup.-1) 0.0940 0.1375 0.1283 0.0754
T.sub.1/2 (hr) 7.38 5.04 5.40 9.20 V.sub.d (L/kg) 2.99 2.22 4.25
5.77 CL (L/ 0.281 0.305 0.545 0.435 hr/kg) V.sub.SS (L/kg) 0.228
0.242 1.44 1.09 K.sub.e: elimination rate constant T.sub.1/2:
elimination half-life V.sub.d: volume of distribution CL: plasma
clearance V.sub.SS: volume of distribution at steady state
Both the C.sub.max and AUC.sub.0.fwdarw..infin. values following
the i.v. bolus administration of QW8184 were significantly higher
than the corresponding values following the i.v. infusion of
Taxol.RTM.. The terminal T.sub.1/2 of paclitaxel in plasma were
similar for the two treatments. Tissue binding was more extensive
with Taxol.RTM. than QW8184 as indicated from differences in the
volume of distribution at steady state (Vss). No significant
differences in the pharmacokinetic parameters of paclitaxel were
observed between male and female animals.
* * * * *